Vpr protein of human immunodeficiency virus type 1 binds to 14-3-3 proteins and facilitates complex formation with Cdc25C: implications for cell cycle arrest.
Vpr and selected mutants were used in a Saccharomyces cerevisiae two-hybrid screen to identify cellular interactors. We found Vpr interacted with 14-3-3 proteins, a family regulating a multitude of proteins in the cell. Vpr mutant R80A, which is inactive in cell cycle arrest, did not interact with 14-3-3. 14-3-3 proteins regulate the G(2)/M transition by inactivating Cdc25C phosphatase via binding to the phosphorylated serine residue at position 216 of Cdc25C. 14-3-3 overexpression in human cells synergized with Vpr in the arrest of cell cycle. Vpr did not arrest efficiently cells not expressing 14-3-3sigma. This indicated that a full complement of 14-3-3 proteins is necessary for optimal Vpr function on the cell cycle. Mutational analysis showed that the C-terminal portion of Vpr, known to harbor its cell cycle-arresting activity, bound directly to the C-terminal part of 14-3-3, outside of its phosphopeptide-binding pocket. Vpr expression shifted localization of the mutant Cdc25C S216A to the cytoplasm, indicating that Vpr promotes the association of 14-3-3 and Cdc25C, independently of the presence of serine 216. Immunoprecipitations of cell extracts indicated the presence of triple complexes (Vpr/14-3-3/Cdc25C). These results indicate that Vpr promotes cell cycle arrest at the G(2)/M phase by facilitating association of 14-3-3 and Cdc25C independently of the latter's phosphorylation status.
Kino
Tomoshige
T
Human Retrovirus Section, Center for Cancer Research, National Cancer Institute-Frederick, Bldg. 535, Rm. 210, Frederick, MD 21702-1201, USA.
Gragerov
Alexander
A
Valentin
Antonio
A
Tsopanomihalou
Maria
M
Ilyina-Gragerova
Galina
G
Erwin-Cohen
Rebecca
R
Chrousos
George P
GP
Pavlakis
George N
GN
D048948:14-3-3 Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002453:Cell Cycle; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D016336:Gene Products, vpr / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015497:HIV-1 / Q000235:genetics / Q000378:metabolism* / Q000472:pathogenicity*; D006367:HeLa Cells; D006801:Humans; D066298:In Vitro Techniques; D046912:Multiprotein Complexes; D011485:Protein Binding; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D020798:Two-Hybrid System Techniques; D020687:cdc25 Phosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D054325:vpr Gene Products, Human Immunodeficiency Virus
D048948:14-3-3 Proteins; D018797:Cell Cycle Proteins; D016336:Gene Products, vpr; D046912:Multiprotein Complexes; D011994:Recombinant Proteins; D054325:vpr Gene Products, Human Immunodeficiency Virus; C517009:CDC25C protein, human; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1; D020687:cdc25 Phosphatases