title: Sulfonation and phosphorylation of regions of the dioxin receptor susceptible to methionine modifications.
abstract: Tagged murine dioxin receptor was purified from mammalian cells, digested with trypsin, and analyzed by capillary HPLC-MALDI-TOF/TOF-MS and -MS/MS. Several chromatographically distinct semitryptic peptides matching two regions spanning residues Glu(409)-Arg(424) and Ser(547)-Arg(555) of the dioxin receptor were revealed by de novo sequencing. Methionine residues at 418 and 548 were detected in these peptides as either unmodified or modified by moieties of 16 (oxidation) or 57 amu (S-carboxamidomethylation) or in a form corresponding to degradative removal of 105 amu from the S-carboxamidomethylated methionine. MS/MS spectra revealed that the peptides containing modified methionine residues also existed in forms with a modification of +80 amu on serine residues 411, 415, and 547. The MS/MS spectra of these peptide ions also revealed diagnostic neutral loss fragment ions of 64, 98, and/or 80 amu, and in some instances combinations of these neutral losses were apparent. Taken together, these data indicated that serines 411 and 547 of the dioxin receptor were sulfonated and serine 415 was phosphorylated. Separate digests of the dioxin receptor were prepared in H(2)(16)O and H(2)(18)O, and enzymatic dephosphorylation was subsequently performed on the H(2)(16)O digest only. The digests were mixed in equal proportions and analyzed by capillary HPLC-MALDI-TOF/TOF-MS and -MS/MS. This strategy confirmed assignment of sulfonation as the cause of the +80-amu modifications on serines 411 and 547 and phosphorylation as the predominant cause of the +80-amu modification of serine 415. The relative quantitation of phosphorylation and sulfonation enabled by this differential phosphatase strategy also suggested the presence of sulfonation on a serine other than residue 411 within the sequence spanning Glu(409)-Arg(424). This represents the first description of post-translational sulfonation sites and identification of a new phosphorylation site of the latent dioxin receptor. Furthermore this is only the second report of serine sulfonation of eukaryotic proteins. Mutagenesis studies are underway to assess the functional consequences of these modifications.
authors.affiliation: Protein Discovery Centre, Queensland Institute of Medical Research, P. O. Royal Brisbane Hospital, Herston, Queensland 4029, Australia.
authors.forename: Keyur A
authors.initials: KA
authors.lastname: Dave
authors.forename: Fiona
authors.initials: F
authors.lastname: Whelan
authors.forename: Colleen
authors.initials: C
authors.lastname: Bindloss
authors.forename: Sebastian G B
authors.initials: SG
authors.lastname: Furness
authors.forename: Anne
authors.initials: A
authors.lastname: Chapman-Smith
authors.forename: Murray L
authors.initials: ML
authors.lastname: Whitelaw
authors.forename: Jeffrey J
authors.initials: JJ
authors.lastname: Gorman
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D006801:Humans; D008715:Methionine / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D010455:Peptides / Q000737:chemistry; D010710:Phosphates / Q000378:metabolism; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D018336:Receptors, Aryl Hydrocarbon / Q000737:chemistry / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013455:Sulfur / Q000378:metabolism*; D014357:Trypsin / Q000378:metabolism
chemical_list: D050505:Mutant Proteins; D010455:Peptides; D010710:Phosphates; D018336:Receptors, Aryl Hydrocarbon; D010768:Phosphoserine; D013455:Sulfur; D008715:Methionine; D010749:Phosphoprotein Phosphatases; D014357:Trypsin