Human immunodeficiency virus type 1 Nef selectively associates with a catalytically active subpopulation of p21-activated kinase 2 (PAK2) independently of PAK2 binding to Nck or beta-PIX

We have recently identified the Nef-associated serine-threonine kinase (NAK ) as the p21-activated kinase 2 (PAK2). Here we have taken advantage of the possibility to manipulate the functional properties of NAK by transfecting PAK2 cDNA or its mutant derivatives in order to further characterize the N ef-NAK complex. To exclude the possibility that some Nef variants might int eract with PAK1 instead of PAK2, we also examined the identity of NAK compl exed with divergent human immunodeficiency virus type 1 HIV-1 Nef proteins. All tested Nef proteins, including SF2, NL4-3, BH10, and HAN-2, associated with PAK2 but not with PAK1. By exchanging different regions between these two PAK proteins, the selective ability of PAK2 to associate with Nef coul d be mapped to the carboxy-terminal part of its regulatory domain. Binding of PAK2 with the adapter protein Nck or beta -PIX was found to be dispensab le for the assembly of the Nef-PAK2 complex, whereas an intact Cdc42-Rac1 i nteractive binding motif was required. Most importantly, we found that NAK represented a distinct subpopulation of the total cellular PAK2 characteriz ed by a high specific kinase activity. Thus, although only a small fraction of cellular PAK2 could be found in complex with Nef, NAK represented a maj or part of cellular PAK2 activity.