Human-immunodeficiency-virus-type-1-encoded Vpu protein is phosphorylated by casein kinase II.
Vpu as a human-immunodeficiency-virus-type-1-encoded 81-amino-acid integral-membrane protein was expressed in Escherichia coli using the inducible ptrc promoter of an ATG fusion vector. Recombinant Vpu is associated with membranes of E. coli and could be partially solubilized by detergents. Recombinant Vpu was phosphorylated in vitro with purified porcine casein kinase II (CKII) as well as with a CKII-related protein kinase found in cytoplasmic extracts of human and hamster cells. Recombinant Vpu associated with E. coli membranes has turned out to be the best substrate for in vitro phosphorylation with CKII. This reaction can be inhibited by heparin and the ATP analogue 5,6-dichloro-1-(beta-D-ribofuranosyl)benzimidazole (DRB), both known to be potent inhibitors of CKII. Radiolabelled gamma ATP and gamma GTP were used as phosphate donors in vitro phosphorylation of recombinant Vpu. In vivo phosphorylation of Vpu in HIV-1-infected H9 cells was also inhibited by DRB. We concluded therefrom that the Vpu protein is phosphorylated by the ubiquitous CKII in HIV-1-infected human host cells. Two seryl residues in the sequence of Vpu (position 52 and 56) correspond to the consensus S/TXXD/E for CKII. These potential phosphorylation sites are located within a well-conserved dodecapeptide of Vpu (residues 47-58), which is found in different HIV-1 strains as well as in a Vpu-like protein of SIVCPZ. Monoclonal and polyclonal antibodies directed against two different epitopes of Vpu were used for immunoprecipitation of Vpu from HIV-1-infected cells and for detection of Vpu in Western blot analyses. Vpu from HIV-1-infected cells as well as recombinant Vpu expressed in E. coli were determined by SDS/PAGE using 6 M urea to be 9 kDa, which corresponds to the calculated molecular mass of Vpu.
Schubert
U
U
Institut für Medizinische Immunologie, Medizinische Fakultät (Charité), Humboldt-Universität zu Berlin, Federal Republic of Germany.
Schneider
T
T
Henklein
P
P
Hoffmann
K
K
Berthold
E
E
Hauser
H
H
Pauli
G
G
Porstmann
T
T
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000096:biosynthesis; D001345:Autoradiography; D015153:Blotting, Western; D047390:Casein Kinase II; D002478:Cells, Cultured; D004004:Dichlororibofuranosylbenzimidazole / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D015497:HIV-1 / Q000378:metabolism*; D054298:Human Immunodeficiency Virus Proteins; D006801:Humans; D007668:Kidney / Q000166:cytology / Q000196:embryology; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011233:Precipitin Tests; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D054334:Viral Regulatory and Accessory Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D000086082:Viroporin Proteins
D000255:Adenosine Triphosphate; D000911:Antibodies, Monoclonal; D047390:Casein Kinase II; D004004:Dichlororibofuranosylbenzimidazole; D006160:Guanosine Triphosphate; D054298:Human Immunodeficiency Virus Proteins; D017346:Protein Serine-Threonine Kinases; D011994:Recombinant Proteins; D054334:Viral Regulatory and Accessory Proteins; C514859:vpu protein, Human immunodeficiency virus 1; D000086082:Viroporin Proteins