title: Identification of a new in vivo phosphorylation site in the cytoplasmic carboxyl terminus of EBV-LMP1 by tandem mass spectrometry.
abstract: Latent membrane protein 1 (LMP1), an oncogenic protein encoded by Epstein-Barr virus (EBV), has been verified to be phosphorylated in vitro by protein casein kinase 2 (CK2). In this study, we characterized the phosphorylation of the carboxyl terminus of LMP1 fused with glutathione-S-transferase (GST-LMP1c) and the FLAG-epitope-tagged LMP1 (F-LMP1) proteins expressed in HEK293T cells. Using a combination of chemical modification and tandem mass spectrometry, we detected the phosphorylation of a tryptic peptide, 191-223 amino acids, in both GST-LMP1c catalysed by CK2 and F-LMP1-expressing cell lines. Serine residues at positions 211 and 215 were determined to be the substrates of CK2 in vitro. Most importantly, the S215 phosphorylation was also detected in F-LMP1-expressing human cell lines. The phosphorylation of S215, which is located in the carboxyl-terminus activation region 1 of LMP1, provides a new insight for investigating the role and modulation of the phosphorylation of LMP1.
authors.affiliation: Department of Biochemistry and Molecular Biology, Chang Gung University, Kwei-shan, Tao-Yuan, Taiwan.
authors.forename: Kun-Yi
authors.initials: KY
authors.lastname: Chien
authors.forename: Yu-Sun
authors.initials: YS
authors.lastname: Chang
authors.forename: Jau-Song
authors.initials: JS
authors.lastname: Yu
authors.forename: Lin-Wen
authors.initials: LW
authors.lastname: Fan
authors.forename: Chien-Wei
authors.initials: CW
authors.lastname: Lee
authors.forename: Lang-Ming
authors.initials: LM
authors.lastname: Chi
mesh_terms: D000595:Amino Acid Sequence; D002384:Catalysis; D002460:Cell Line; D005982:Glutathione Transferase / Q000737:chemistry; D004854:Herpesvirus 4, Human / Q000378:metabolism; D006801:Humans; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D014763:Viral Matrix Proteins / Q000737:chemistry* / Q000378:metabolism
chemical_list: C037662:EBV-associated membrane antigen, Epstein-Barr virus; D011993:Recombinant Fusion Proteins; D014763:Viral Matrix Proteins; D005982:Glutathione Transferase