HIV-1 Nef Recruits the Guanine Exchange Factor Vav1 via an Unexpected Interface into Plasma Membrane Microdomains for Association with PAK2 Activity

Abteilung Virologie, Universitätsklinikum Heidelberg, Heidelberg, Germany; Institute of Medical Technology, University of Tampere, Tampere, Finland; Department of Virology, Haartman Institute, University of Helsinki and Helsinki University Central Hospital, Helsinki, Finland

^* To whom correspondence should be addressed. Email: oliver_fackler{at}med.uni-heidelberg.de.

	Abstract

Alterations of T-cell receptor signaling by HIV-1 Nef involve its association with a highly active subpopulation of p21-activated kinase 2 (PAK2) within a dynamic signalosome assembled in detergent-insoluble membrane microdomains. Nef-PAK2 complexes contain the GTPases Rac and Cdc42 as well as a factor providing guanine nucleotide exchange factor (GEF) activity for Rac/Cdc42. However, the identity of this GEF has remained controversial. Previous studies suggested the association of Nef with at least three independent GEFs, Vav, DOCK2/ELMO1 and Pix. Here we use a broad panel of approaches to address which of these GEFs is involved in the functional interaction of Nef with PAK2 activity. Biochemical fractionation and confocal microscopy revealed that Nef recruits Vav1, but not DOCK2/ELMO1 or Pix, to membrane microdomains. Transient RNAi knock-down, analysis of cell lines defective for expression of Vav1 or DOCK2 as well as use of a Pix binding deficient PAK2 variant confirmed a role for Vav1 but not DOCK2 or Pix for Nef's association with PAK2 activity. Nef-mediated microdomain recruitment of Vav1 occurred independently of the SH3 binding PxxP motif, which is known to connect Nef to many cellular signaling processes. Instead, a recently described protein interaction surface surrounding Nef residue F195 was identified as critical for Nef-mediated raft recruitment of Vav1. These results identify Vav1 as a relevant component of the Nef-PAK2 signalosome and provide a molecular basis for the role of F195 in formation a catalytically active Nef-PAK2 complex.