Nef-mediated lipid raft exclusion of UbcH7 inhibits Cbl activity in T cells to positively regulate signaling.
Lentiviral Nef increases T cell signaling activity, but the molecular nature of the stimulus involved is incompletely described. We explored CD4 T cell lipid raft composition in the presence and absence of Nef. Here, the E2 ubiquitin-conjugating enzyme UbcH7, which acts in conjunction with c-Cbl, is absent from lipid rafts. This Nef-mediated exclusion is associated with failure of ubiquitination of activated Vav. In the presence of Nef, lipid raft Cdc42 is activated and forms a ternary complex between the c-Cbl-interacting protein p85Cool-1/betaPix and c-Cbl, displacing UbcH7 from rafts. Suppression of p85Cool-1/betaPix expression restores UbcH7 raft localization and Vav ubiquitination and diminishes Cdc42 activity. Moreover, p85Cool-1/betaPix knockdown attenuates HIV replication. Thresholds for activation of signaling involve the intricate balance of positive and negative regulators. Here we provide evidence for Nef disruption of a negative regulator of T cell signaling in promoting HIV replication.
Simmons
Alison
A
MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Headington, Oxford OX3 9DS, United Kingdom. alison.simmons@molecular-medicine.oxford.ac.uk
Gangadharan
Bevin
B
Hodges
Ashleigh
A
Sharrocks
Katherine
K
Prabhakar
Sripadi
S
García
Angel
A
Dwek
Raymond
R
Zitzmann
Nicole
N
McMichael
Andrew
A
D015153:Blotting, Western; D015496:CD4-Positive T-Lymphocytes / Q000166:cytology / Q000276:immunology*; D018797:Cell Cycle Proteins / Q000378:metabolism; D002460:Cell Line; D004797:Enzyme-Linked Immunosorbent Assay; D015693:Gene Products, nef / Q000276:immunology / Q000378:metabolism*; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism; D006678:HIV; D006801:Humans; D047468:Immunoprecipitation; D021962:Membrane Microdomains / Q000276:immunology / Q000378:metabolism*; D018613:Microscopy, Confocal; D050721:Proto-Oncogene Proteins c-cbl / Q000276:immunology / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics; D064067:Rho Guanine Nucleotide Exchange Factors; D015398:Signal Transduction / Q000276:immunology*; D044763:Ubiquitin-Conjugating Enzymes / Q000276:immunology / Q000378:metabolism*; D014779:Virus Replication / Q000276:immunology*; D020764:cdc42 GTP-Binding Protein / Q000378:metabolism; D054311:nef Gene Products, Human Immunodeficiency Virus
D018797:Cell Cycle Proteins; D015693:Gene Products, nef; D020662:Guanine Nucleotide Exchange Factors; D034741:RNA, Small Interfering; D064067:Rho Guanine Nucleotide Exchange Factors; D054311:nef Gene Products, Human Immunodeficiency Virus; C079437:UBE2L3 protein, human; D044763:Ubiquitin-Conjugating Enzymes; D050721:Proto-Oncogene Proteins c-cbl; D020764:cdc42 GTP-Binding Protein; C067419:CBL protein, human