Nef associates with p21-activated kinase 2 in a p21-GTPase-dependent dynamic activation complex within lipid rafts.

We have previously reported that Nef specifically interacts with a small but highly active subpopulation of p21-activated kinase 2 (PAK2). Here we show that this is due to a transient association of Nef with a PAK2 activation complex within a detergent-insoluble membrane compartment containing the lipid raft marker GM1. The low abundance of this Nef-associated kinase (NAK) complex was found to be due to an autoregulatory mechanism. Although activation of PAK2 was required for assembly of the NAK complex, catalytic activity of PAK2 also promoted dissociation of this complex. Testing different constitutively active PAK2 mutants indicated that the conformation associated with p21-mediated activation rather than kinase activity per se was required for PAK2 to become NAK. Although association with PAK2 is one of the most conserved properties of Nef, we found that the ability to stimulate PAK2 activity differed markedly among divergent Nef alleles, suggesting that PAK2 association and activation are distinct functions of Nef. However, mutations introduced into the p21-binding domain of PAK2 revealed that p21-GTPases are involved in both of these Nef functions and, in addition to promoting PAK2 activation, also help to physically stabilize the NAK complex.