SH3-Domain binding function of HIV-1 Nef is required for association with a PAK-related kinase

HIV-1 Nef has previously been shown to bind to Src homology-3 (SH3) domains of a subset of Src family tyrosine kinases. In addition, Nef has been reported to coprecipitate with a serine/threonine kinase activity termed NAK (for Nef-associated kinase). The identity of NAK remains uncertain, but it has been suggested to represent a novel member of the p21-activated kinase (PAK) family. We report here that NAK autophosphorylation is increased not only by an activated form of the p21-family GTPase cdc42 but also by a plasma membrane-targeted fragment of the adapter protein Nck, thus providing further evidence that NAK is related to PAKs. A detailed structure-based mutational analysis of Nef revealed that all amino acid changes that inhibited the Nef/Hck-SH3 interaction, as measured by surface-plasmon resonance, also abolished coprecipitation of NAK. As PAK family proteins do not contain SH3 domains, these observations are best explained by a protein complex in which Nef, NAK, and an SH3-protein all contact each other. In addition, a number of conserved amino acids in Nef that are not involved in SH3 binding were also found to be crucial for association with NAK. Molecular modeling suggests that these residues are involved in formation of an adjacent binding surface for NAK or another critical component of the NAK/Nef complex.