Lentivirus Nef specifically activates Pak2

Nef proteins from human immunodeficiency virus type 1 (HIV-1) and simian im munodeficiency virus (SM have been found to associate with an active cellul ar serine/threonine kinase designated Nef-associated kinase (Nak). The exac t identity of Nak remains controversial, with two recent studies indicating that Nak may be either Pak1 or Pak2. In this study, we investigated the hy pothesis that such discrepancies arise from the use of different Nef allele s or different cell types by individual investigators. We first confirm tha t Pak2 but not Pak1 is cleaved by caspase 3 in vitro and then demonstrate t hat Nak is caspase 3 sensitive, regardless of Nef allele or cell type used. We tested nef alleles from three lentiviruses (HIV-1 SF2, HIV-1 NL4-3, and SIVmac239) and used multiple cell lines of myeloid, lymphoid, and nonhemat opoietic origin to evaluate the identity of Nak. We demonstrate that ectopi cally expressed Pak2 can substitute for Nak, while ectopically expressed Pa k1 cannot. We then show that Nef specifically mediates the robust activatio n of ectopically expressed Pak2, directly demonstrating that Nef regulates Pak2 activity and does not merely associate with activated Pak2. We report that most of the active Pak2 is found bound to Nef, although a fraction is not. In contrast, only a small amount of Nef is found associated with Pak2. We conclude that Nak is Pak2 and that Nef specifically mediates Pak2 activ ation in a low-abundance complex. These results will facilitate both the el ucidation of the role of Nef in pathogenesis and the development of specifi c inhibitors of this highly conserved function of Nef.