|
BMC Chemical Biology 2012
HIV-1 Nef interaction influences the ATP-binding site of the Src-family kinase, HckAbstract: To test this hypothesis, we engineered a "gatekeeper" mutant of Hck with enhanced sensitivity to the pyrazolopyrimidine tyrosine kinase inhibitor, NaPP1. We also modified the RT loop of the Hck SH3 domain to enhance interaction of the kinase with Nef. This modification stabilized Nef:Hck interaction in solution-based kinase assays, as a way to mimic the more stable association that likely occurs at cellular membranes. Introduction of the modified RT loop rendered Hck remarkably more sensitive to activation by Nef, and led to a significant decrease in the Km for ATP as well as enhanced inhibitor potency.These observations suggest that stable interaction with Nef may induce Src-family kinase active site conformations amenable to selective inhibitor targeting.Nef is an HIV-1 accessory protein that facilitates virus infectivity, replication, and immune evasion [1-3]. In non-human primate models of AIDS, high-titer viral replication and development of AIDS-like disease requires an intact Nef gene [4]. Long-term non-progressive HIV infection in humans is also associated with Nef-defective HIV isolates in some cases [5,6]. Complementary in vivo studies have shown that directed expression of Nef alone to HIV target cells induces an AIDS-like syndrome in transgenic mice [7-9]. Taken together, these studies underscore the importance of HIV-1 Nef in AIDS pathogenesis.Nef is not known to exhibit any intrinsic enzymatic activity. Instead, Nef interacts with multiple host cell signaling pathways to enhance HIV-1 replication and promote AIDS progression [10]. Previous work from our group has identified members of the Src kinase family as direct Nef effectors [11-15]. This kinase family includes Hck, a Src-family member expressed in macrophages, which are a critical HIV target cell type and viral reservoir. Nef interacts with the Hck SH3 domain, leading to constitutive Hck activation that may contribute to macrophage survival, MHC-I downregulation and M-tropic HIV replication [11,1
|