Nef-mediated lipid raft exclusion of UbcH7 inhibits Cbl activity in T cells to positively regulate signaling.

Simmons A, Gangadharan B, Hodges A, Sharrocks K, Prabhakar S, García A, Dwek R, Zitzmann N, McMichael A

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.

Keywords:

Blotting, Western

,

CD4-Positive T-Lymphocytes

,

Cell Cycle Proteins

,

Cell Line

,

Enzyme-Linked Immunosorbent Assay

,

Gene Products, nef

,

Guanine Nucleotide Exchange Factors

,

HIV

,

Humans

,

Immunoprecipitation

,

Membrane Microdomains

,

Microscopy, Confocal

,

Proto-Oncogene Proteins c-cbl

,

RNA, Small Interfering

,

Rho Guanine Nucleotide Exchange Factors

,

Signal Transduction

,

Ubiquitin-Conjugating Enzymes

,

Virus Replication

,

cdc42 GTP-Binding Protein

,

nef Gene Products, Human Immunodeficiency Virus