title: The PA influenza virus polymerase subunit is a phosphorylated protein.
abstract: The induction of proteolysis by expression of the influenza virus PA polymerase subunit is the only biochemical activity ascribed to this protein. In the course of studying viral protein synthesis by two-dimensional gel electrophoresis, we observed the existence of several PA isoforms with different isoelectric points. These isoforms were also present when the PA gene was singly expressed in three different expression systems, indicating that a cellular activity is responsible for its post-translational modification. In vivo labelling with [32P]orthophosphate, followed by two-dimensional gel electrophoresis, clearly demonstrated the incorporation of phosphate into the PA molecule. Phosphoserine and phosphothreonine epitopes were present in PA, while phosphotyrosine residues were absent, as tested by immunoblotting with specific antibodies. These facts, as well as the presence of multiple consensus sites for casein kinase II (CKII) phosphorylation, prompted us to test the involvement of this kinase in PA covalent modification. PA protein purified by immunoprecipitation could be specifically labelled by the catalytic alpha subunit of human CKII, which was expressed and purified from bacteria. Collectively, these data demonstrate that the PA subunit of the influenza virus RNA polymerase is a phosphoprotein.
authors.affiliation: Centro Nacional de Biotecnología (CSIC), Cantoblanco, Madrid, Spain.
authors.forename: J J
authors.initials: JJ
authors.lastname: Sanz-Ezquerro
authors.forename: J
authors.initials: J
authors.lastname: Fernández Santarén
authors.forename: T
authors.initials: T
authors.lastname: Sierra
authors.forename: T
authors.initials: T
authors.lastname: Aragón
authors.forename: J
authors.initials: J
authors.lastname: Ortega
authors.forename: J
authors.initials: J
authors.lastname: Ortín
authors.forename: G L
authors.initials: GL
authors.lastname: Smith
authors.forename: A
authors.initials: A
authors.lastname: Nieto
mesh_terms: D000818:Animals; D015153:Blotting, Western; D019556:COS Cells; D047390:Casein Kinase II; D004268:DNA-Binding Proteins / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional; D010766:Phosphorylation; D010768:Phosphoserine / Q000032:analysis; D010769:Phosphothreonine / Q000032:analysis; D011499:Protein Processing, Post-Translational; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D012324:RNA-Dependent RNA Polymerase; D014162:Transfection; D014764:Viral Proteins / Q000737:chemistry / Q000378:metabolism*
chemical_list: D004268:DNA-Binding Proteins; C061290:PA protein, influenza viruses; D014764:Viral Proteins; D010769:Phosphothreonine; D010768:Phosphoserine; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases; D012324:RNA-Dependent RNA Polymerase; D012321:DNA-Directed RNA Polymerases