title: Phosphorylation of the chromatin binding domain of KSHV LANA.
abstract: The Kaposi sarcoma associated herpesvirus (KSHV) latency associated nuclear antigen (LANA) is expressed in all KSHV associated malignancies and is essential for maintenance of KSHV genomes in infected cells. To identify kinases that are potentially capable of modifying LANA, in vitro phosphorylation assays were performed using an Epstein Barr virus plus LANA protein microarray and 268 human kinases purified in active form from yeast. Interestingly, of the Epstein-Barr virus proteins on the array, the EBNA1 protein had the most similar kinase profile to LANA. We focused on nuclear kinases and on the N-terminus of LANA (amino acids 1-329) that contains the LANA chromatin binding domain. Sixty-three nuclear kinases phosphorylated the LANA N-terminus. Twenty-four nuclear kinases phosphorylated a peptide covering the LANA chromatin binding domain (amino acids 3-21). Alanine mutations of serine 10 and threonine 14 abolish or severely diminish chromatin and histone binding by LANA. However, conversion of these residues to the phosphomimetic glutamic acid restored histone binding suggesting that phosphorylation of serine 10 and threonine 14 may modulate LANA function. Serine 10 and threonine 14 were validated as substrates of casein kinase 1, PIM1, GSK-3 and RSK3 kinases. Short-term treatment of transfected cells with inhibitors of these kinases found that only RSK inhibition reduced LANA interaction with endogenous histone H2B. Extended treatment of PEL cell cultures with RSK inhibitor caused a decrease in LANA protein levels associated with p21 induction and a loss of PEL cell viability. The data indicate that RSK phosphorylation affects both LANA accumulation and function.
authors.affiliation: High Throughput Biology Center, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.
authors.forename: Crystal
authors.initials: C
authors.lastname: Woodard
authors.forename: Meir
authors.initials: M
authors.lastname: Shamay
authors.forename: Gangling
authors.initials: G
authors.lastname: Liao
authors.forename: Jian
authors.initials: J
authors.lastname: Zhu
authors.forename: Ai Na
authors.initials: AN
authors.lastname: Ng
authors.forename: Renfeng
authors.initials: R
authors.lastname: Li
authors.forename: Rob
authors.initials: R
authors.lastname: Newman
authors.forename: Hee-Sool
authors.initials: HS
authors.lastname: Rho
authors.forename: Jianfei
authors.initials: J
authors.lastname: Hu
authors.forename: Jun
authors.initials: J
authors.lastname: Wan
authors.forename: Jiang
authors.initials: J
authors.lastname: Qian
authors.forename: Heng
authors.initials: H
authors.lastname: Zhu
authors.forename: S Diane
authors.initials: SD
authors.lastname: Hayward
mesh_terms: D000595:Amino Acid Sequence; D000956:Antigens, Viral / Q000737:chemistry* / Q000378:metabolism*; D001665:Binding Sites; D047389:Casein Kinase I / Q000037:antagonists & inhibitors / Q000378:metabolism; D002460:Cell Line; D002843:Chromatin / Q000378:metabolism; D005656:Fungal Proteins; D038362:Glycogen Synthase Kinase 3 / Q000037:antagonists & inhibitors / Q000378:metabolism; D057809:HEK293 Cells; D004854:Herpesvirus 4, Human; D019288:Herpesvirus 8, Human / Q000378:metabolism*; D006657:Histones / Q000378:metabolism; D006801:Humans; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D009687:Nuclear Proteins / Q000737:chemistry* / Q000378:metabolism*; D010766:Phosphorylation; D054730:Protein Interaction Domains and Motifs; D011494:Protein Kinases / Q000378:metabolism*; D038744:Ribosomal Protein S6 Kinases, 90-kDa / Q000037:antagonists & inhibitors / Q000378:metabolism*; D012514:Sarcoma, Kaposi / Q000821:virology
chemical_list: D000956:Antigens, Viral; D002843:Chromatin; D005656:Fungal Proteins; D006657:Histones; D009687:Nuclear Proteins; C109540:latency-associated nuclear antigen; D011494:Protein Kinases; D047389:Casein Kinase I; D038744:Ribosomal Protein S6 Kinases, 90-kDa; C069330:ribosomal protein S6 kinase, 90kDa, polypeptide 3; D020928:Mitogen-Activated Protein Kinases; C432694:Pim1 protein, Pichia pastoris; D038362:Glycogen Synthase Kinase 3