       Document 0218
 DOCN  M9440218
 TI    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.
 DT    9404
 AU    Schubert U; Henklein P; Boldyreff B; Wingender E; Strebel K; Porstmann
       T; Institut fur Medizinische Immunologie, Medizinische Fakultat;
       (Charite), Humboldt-Universitat zu Berlin, Germany.
 SO    J Mol Biol. 1994 Feb 11;236(1):16-25. Unique Identifier : AIDSLINE
       MED/94149711
 AB    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.
 DE    Amino Acid Sequence  Base Sequence  DNA Primers  Gene Products,
       vpu/BIOSYNTHESIS/*CHEMISTRY/*METABOLISM  Human  HIV-1/*METABOLISM
       Molecular Sequence Data  Mutagenesis, Site-Directed  Phosphorylation
       Polymerase Chain Reaction  Protein Kinases/*METABOLISM  *Protein
       Structure, Secondary  Recombinant
       Proteins/BIOSYNTHESIS/CHEMISTRY/METABOLISM  Reticulocytes/METABOLISM
       *Serine  Support, Non-U.S. Gov't  Translation, Genetic  JOURNAL ARTICLE

       SOURCE: National Library of Medicine.  NOTICE: This material may be
       protected by Copyright Law (Title 17, U.S.Code).

