Abstract
BackgroundNef is an HIV-1 accessory protein essential for viral replication and AIDS progression. Nef interacts with a multitude of host cell signaling partners, including members of the Src kinase family. Nef preferentially activates Hck, a Src-family kinase (SFK) strongly expressed in macrophages and other HIV target cells, by binding to its regulatory SH3 domain. Recently, we identified a series of kinase inhibitors that preferentially inhibit Hck in the presence of Nef. These compounds also block Nef-dependent HIV replication, validating the Nef-SFK signaling pathway as an antiretroviral drug target. Our findings also suggested that by binding to the Hck SH3 domain, Nef indirectly affects the conformation of the kinase active site to favor inhibitor association.ResultsTo 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.ConclusionsThese observations suggest that stable interaction with Nef may induce Src-family kinase active site conformations amenable to selective inhibitor targeting.
Highlights
Introduction ofSH3-High Affinity RT loop (HART) sensitizes near full-length mouse Hck (mHck) to activation by Nef To determine the impact of the SH3-HART modification in the context of near full-length Hck, we introduced the HART coding sequence into mHck-TA
A cDNA clone for mouse Hck was modified at its gatekeeper position (Thr338) with alanine, the Cterminal tail was changed to the autoregulatory sequence, Tyr-Glu-Glu-Ile (YEEI), and the N-terminal unique domain was replaced with a hexahistidine tag as described previously for human Hck [27]
Expression and characterization of a mouse Hck gatekeeper mutant Previous studies have shown that Nef forms a stable complex with Hck in cells, and that this interaction results in constitutive Hck activation [19]
Summary
Introduction ofSH3-HART sensitizes near full-length mHck to activation by Nef To determine the impact of the SH3-HART modification in the context of near full-length Hck, we introduced the HART coding sequence into mHck-TA. Because the HART sequence strongly enhanced interaction of the isolated SH3 domain with Nef, we predicted that mHck-TAHART should be activated at lower concentrations of Nef than mHck-TA with a wild-type SH3 domain. To test this hypothesis, we conducted in vitro kinase assays over a range of kinase:Nef ratios for both Nef variants. Nef preferentially activates Hck, a Src-family kinase (SFK) strongly expressed in macrophages and other HIV target cells, by binding to its regulatory SH3 domain. Complementary in vivo studies have shown that directed expression of Nef alone to HIV target cells induces an AIDS-like syndrome in transgenic mice [7,8,9]. Nef interacts with multiple host cell signaling pathways to enhance HIV-1 replication and
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