title: Human immunodeficiency virus type 1 Nef activates p21-activated kinase via recruitment into lipid rafts.
abstract: The Nef protein of human immunodeficiency virus type 1 is an important factor in AIDS pathogenesis. In addition to downregulating CD4 and major histocompatibility complex class I molecules from the cell surface, as well as increasing virion infectivity, Nef triggers activation of the T-cell receptor (TCR) cascade to facilitate virus spread. Signaling pathways that are induced by Nef have been identified; however, it is unclear how and in which subcellular compartment Nef triggers signaling. Nef recruits a multiprotein complex to activate the cellular Pak kinase that mediates downstream effector functions. Since a subpopulation of Nef is present in detergent-insoluble microdomains (lipid rafts) from where physiological TCR signaling is initiated, we tested whether lipid rafts are instrumental for Nef-mediated Pak activation. In flotation analysis, Nef-associated Pak activity exclusively fractionated with lipid rafts. Activation of Pak in the presence of Nef coincided with lipid raft recruitment of the kinase, which was otherwise excluded from detergent-insoluble microdomains. Experimental solubilization of lipid rafts interfered with the association of Pak activity with Nef. To analyze the importance of the raft localization for Nef function more rigorously, we generated a palmitoylated Nef (PalmNef). PalmNef was highly enriched in lipid rafts and associated with significantly higher levels of Pak activity than Nef. Notably, activation of Pak by its physiological activators, Cdc42 and Rac, also occurred in lipid rafts and required raft integrity. Together, these data suggest that Nef induces signal transduction via the recruitment of a signaling machinery including Pak into lipid rafts, thereby mimicking a physiological cellular mechanism to initiate the TCR cascade.
authors.affiliation: Abteilung Virologie, Universitätsklinikum Heidelberg, D-69120 Heidelberg, Germany.
authors.forename: Ellen
authors.initials: E
authors.lastname: Krautkrämer
authors.forename: Simone I
authors.initials: SI
authors.lastname: Giese
authors.forename: Judith E
authors.initials: JE
authors.lastname: Gasteier
authors.forename: Walter
authors.initials: W
authors.lastname: Muranyi
authors.forename: Oliver T
authors.initials: OT
authors.lastname: Fackler
mesh_terms: D002460:Cell Line; D004789:Enzyme Activation; D015693:Gene Products, nef / Q000235:genetics / Q000502:physiology*; D015497:HIV-1 / Q000235:genetics / Q000472:pathogenicity* / Q000502:physiology*; D006801:Humans; D019169:Jurkat Cells; D021962:Membrane Microdomains / Q000201:enzymology* / Q000821:virology*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011948:Receptors, Antigen, T-Cell / Q000378:metabolism; D015398:Signal Transduction; D013601:T-Lymphocytes / Q000502:physiology / Q000821:virology; D020764:cdc42 GTP-Binding Protein / Q000378:metabolism; D054311:nef Gene Products, Human Immunodeficiency Virus; D054462:p21-Activated Kinases; D020744:rac GTP-Binding Proteins / Q000378:metabolism
chemical_list: D015693:Gene Products, nef; D011948:Receptors, Antigen, T-Cell; D054311:nef Gene Products, Human Immunodeficiency Virus; C515907:PAK2 protein, human; D017346:Protein Serine-Threonine Kinases; D054462:p21-Activated Kinases; D020764:cdc42 GTP-Binding Protein; D020744:rac GTP-Binding Proteins