A well-conserved feature of HIV-1 and SIV Nef is the interaction with and activation of the human p21-activated kinase2 (PAK2). The conservation of this interaction in other species and its significance for Nef pathogenesis in vivo are poorly documented. In the present study, we measured these parameters in Nef expressing thymocytes, macrophages and dendritic cells of a transgenic (Tg) mouse model of AIDS (CD4C/HIV). We found that Nef binds to and activates PAK2, but not PAK1 and 3, in these three cell subsets. Nef associates with only a small fraction of PAK2. The Nef-PAK2 complex also comprises b-PIX/COOL. The impact of the Nef PAK2 association on disease development was also analyzed in Tg mice expressing 10 different Nef mutant alleles. CD4C/HIV Tg mice-expressing Nef alleles defective in Nef-PAK2 association [P69A, P72A/P75A, RR105/106AA, r56-66 or G2A (myristoylation site)] failed to develop disease of the non-lymphoid organs (kidneys and lungs). Among these, only Tg mice expressing NefP69A and NefG2A showed some depletion of CD4+ T cells, although a downregulation of the CD4 surface protein was documented in all these Tg lines, except those expressing Nefr56-66. Among other Tg mice expressing Nef mutants having conserved the Nef-PAK2 association (RD35AA, D174K, P147A/P150A, r8-17, r25 65), only Tg mice expressing Nefr8-17 develop kidney and lung diseases, but all showed partial CD4+ T cell depletion despite some being defective for CD4 downregulation (RD35AA, D174K). Therefore, Nef can activate murine PAK2 and associate with a small fraction of it, as in human cells. Such activation and binding of PAK2 is clearly not sufficient, but may be required to induce a multi organ AIDS like disease in Tg mice.