title: The human immunodeficiency virus type 1 encoded Vpu protein is phosphorylated by casein kinase-2 (CK-2) at positions Ser52 and Ser56 within a predicted alpha-helix-turn-alpha-helix-motif.
abstract: The human immunodeficiency virus type 1 (HIV-1) encoded Vpu is a small integral membrane phosphoprotein that functions in the enhancement of viral particle release and has more recently been shown to cause degradation of CD4 at the endoplasmic reticulum. We have demonstrated earlier that Vpu is phosphorylated by the ubiquitous casein kinase-2 (CK-2) in HIV-1 infected cells. The phosphoacceptor sites targeted by CK-2 in Vpu, however, have not been demonstrated and it was unclear whether Vpu was phosphorylated at one or more of its four serine residues. In this study we characterized the CK-2 phosphoacceptor sites in Vpu using recombinant CK-2 for in vitro phosphorylation of recombinant Vpu protein as well as synthetic peptides of Vpu. Phosphorylation of both Ser52 and Ser56 was demonstrated by in vitro phosphorylation using three 54-residue peptides comprising the entire hydrophilic part of Vpu and containing single serine to asparagine transitions in either position 52 or 56. The Km values of CK-2 to these peptides were established, revealing a preferential phosphorylation of Ser56. The Km values are: Ser56 = 31 microM; Ser 52 = 156 microM; wild type = 27 microM. In addition, we studied phosphorylation of Vpu by endogenous CK-2 following in vitro translation in rabbit reticulocyte lysate of wild-type Vpu or a mutant, Vpum2/6, carrying serine to asparagine changes at amino acid positions 52 and 56. The in vivo phosphorylation of Vpu was studied in transiently transfected human embryonic kidney (293) cells. In this system, the mutant Vpum2/6 was not phosphorylated, indicating that the seryl residues of Vpu at amino acid positions 52 and 56, but not those at positions 23 and 61, are phosphorylated by CK-2. The two CK-2 phosphorylation sites are conserved in all known Vpu sequences and represent the consensus Ser52GlyAsn(Glu/Asp)Ser(Glu/Asp)Gly(Glu/Asp)59. Prediction of the secondary structure revealed a conserved alpha-helix-turn-alpha-helix motif for the hydrophilic C-terminal part of Vpu. A structural model for Vpu is proposed in which the membrane anchor precedes a region comprising two amphipathic alpha-helices of opposed polarity, joined by a strongly acidic turn that protrudes into the cytoplasm and contains the CK-2 phosphorylation sites. Possible functional and structural homologies of Vpu to the membrane channel-forming M2 protein of influenza A viruses are discussed.
authors.affiliation: Institut für Medizinische Immunologie, Medizinische Fakultät (Charité), Humboldt-Universität zu Berlin, Germany.
authors.forename: U
authors.initials: U
authors.lastname: Schubert
authors.forename: P
authors.initials: P
authors.lastname: Henklein
authors.forename: B
authors.initials: B
authors.lastname: Boldyreff
authors.forename: E
authors.initials: E
authors.lastname: Wingender
authors.forename: K
authors.initials: K
authors.lastname: Strebel
authors.forename: T
authors.initials: T
authors.lastname: Porstmann
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D047388:Casein Kinases; D017931:DNA Primers; D015497:HIV-1 / Q000378:metabolism*; D054298:Human Immunodeficiency Virus Proteins; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D011494:Protein Kinases / Q000378:metabolism*; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D012156:Reticulocytes / Q000378:metabolism; D012694:Serine; D054334:Viral Regulatory and Accessory Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D000086082:Viroporin Proteins
chemical_list: D047388:Casein Kinases; D017931:DNA Primers; D054298:Human Immunodeficiency Virus Proteins; D011494:Protein Kinases; D011994:Recombinant Proteins; D012694:Serine; D054334:Viral Regulatory and Accessory Proteins; C514859:vpu protein, Human immunodeficiency virus 1; D000086082:Viroporin Proteins