Sulfonation and phosphorylation of regions of the dioxin receptor susceptible to methionine modifications

Dave, Keyur A., Whelan, Fiona, Bindloss, Colleen, Furness, Sebastian G. B., Chapman-Smith, Anne, Whitelaw, Murray L. and Gorman, Jeffrey J. (2009) Sulfonation and phosphorylation of regions of the dioxin receptor susceptible to methionine modifications. Molecular & Cellular Proteomics, 8 4: 706-719. doi:10.1074/mcp.M800459-MCP200

Author Dave, Keyur A.
Whelan, Fiona
Bindloss, Colleen
Furness, Sebastian G. B.
Chapman-Smith, Anne
Whitelaw, Murray L.
Gorman, Jeffrey J.
Title Sulfonation and phosphorylation of regions of the dioxin receptor susceptible to methionine modifications
Journal name Molecular & Cellular Proteomics   Check publisher's open access policy
ISSN 1535-9476
Publication date 2009-04-01
Sub-type Article (original research)
DOI 10.1074/mcp.M800459-MCP200
Open Access Status DOI
Volume 8
Issue 4
Start page 706
End page 719
Total pages 14
Place of publication United States
Publisher American Society for Biochemistry and Molecular Biology, Inc.
Language eng
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.
Keyword Aryl-hydrocarbon receptor
Q-Index Code C1
Q-Index Status Provisional Code
Institutional Status UQ

Document type: Journal Article
Sub-type: Article (original research)
Collection: Institute for Molecular Bioscience - Publications
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Created: Thu, 03 Sep 2009, 18:17:42 EST by Mr Andrew Martlew on behalf of Institute for Molecular Bioscience