title: Conserved protein kinases encoded by herpesviruses and cellular protein kinase cdc2 target the same phosphorylation site in eukaryotic elongation factor 1delta.
abstract: Earlier studies have shown that translation elongation factor 1delta (EF-1delta) is hyperphosphorylated in various mammalian cells infected with representative alpha-, beta-, and gammaherpesviruses and that the modification is mediated by conserved viral protein kinases encoded by herpesviruses, including UL13 of herpes simplex virus type 1 (HSV-1), UL97 of human cytomegalovirus, and BGLF4 of Epstein-Barr virus (EBV). In the present study, we attempted to identify the site in EF-1delta associated with the hyperphosphorylation by the herpesvirus protein kinases. Our results are as follows: (i) not only in infected cells but also in uninfected cells, replacement of the serine residue at position 133 (Ser-133) of EF-1delta by alanine precluded the posttranslational processing of EF-1delta, which corresponds to the hyperphosphorylation. (ii) A purified chimeric protein consisting of maltose binding protein (MBP) fused to a domain of EF-1delta containing Ser-133 (MBP-EFWt) is specifically phosphorylated in in vitro kinase assays by purified recombinant UL13 fused to glutathione S-transferase (GST) expressed in the baculovirus system. In contrast, the level of phosphorylation by the recombinant UL13 of MBP-EFWt carrying an alanine replacement of Ser-133 (MBP-EFS133A) was greatly impaired. (iii) MBP-EFWt is also specifically phosphorylated in vitro by purified recombinant BGLF4 fused to GST expressed in the baculovirus system, and the level of phosphorylation of MBP-EFS133A by the recombinant BGLF4 was greatly reduced. (iv) The sequence flanking Ser-133 of EF-1delta completely matches the consensus phosphorylation site for a cellular protein kinase, cdc2, and in vitro kinase assays revealed that purified cdc2 phosphorylates Ser-133 of EF-1delta. (v) As observed with EF-1delta, the casein kinase II beta subunit (CKIIbeta) was specifically phosphorylated by UL13 in vitro, while the level of phosphorylation of CKIIbeta by UL13 was greatly diminished when a serine residue at position 209, which has been reported to be phosphorylated by cdc2, was replaced with alanine. These results indicate that the conserved protein kinases encoded by herpesviruses and a cellular protein kinase, cdc2, have the ability to target the same amino acid residues for phosphorylation. Our results raise the possibility that the viral protein kinases mimic cdc2 in infected cells.
authors.affiliation: Department of Cell Regulation, Medical Research Institute, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo 113-8510, Japan. ykawagu@med.nagoya-u.ac.jp
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Kawaguchi
authors.forename: Kentaro
authors.initials: K
authors.lastname: Kato
authors.forename: Michiko
authors.initials: M
authors.lastname: Tanaka
authors.forename: Mikiko
authors.initials: M
authors.lastname: Kanamori
authors.forename: Yukihiro
authors.initials: Y
authors.lastname: Nishiyama
authors.forename: Yuji
authors.initials: Y
authors.lastname: Yamanashi
mesh_terms: D000818:Animals; D016367:Baculoviridae / Q000235:genetics / Q000378:metabolism; D016203:CDC2 Protein Kinase / Q000378:metabolism*; D019556:COS Cells; D047390:Casein Kinase II; D018259:Herpesvirus 1, Human / Q000201:enzymology / Q000235:genetics / Q000472:pathogenicity; D004854:Herpesvirus 4, Human / Q000201:enzymology / Q000235:genetics / Q000472:pathogenicity; D006801:Humans; D020648:Peptide Elongation Factor 1 / Q000737:chemistry* / Q000378:metabolism*; D010766:Phosphorylation; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012694:Serine / Q000737:chemistry / Q000378:metabolism; D014764:Viral Proteins
chemical_list: D020648:Peptide Elongation Factor 1; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D012694:Serine; D011494:Protein Kinases; C406063:BGLF4 protein, Epstein-Barr virus; C082380:UL13 protein, Simplexvirus; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases; D016203:CDC2 Protein Kinase