HIV-1 Nef Selectively Activates Src Family Kinases Hck, Lyn, and c-Src through Direct SH3 Domain Interaction

Abstract

Nef is an HIV-1 virulence factor that promotes viral pathogenicity by altering host cell signaling pathways. Nef binds several members of the Src kinase family, and these interactions have been implicated in the pathogenesis of HIV/AIDS. However, the direct effect of Nef interaction on Src family kinase (SFK) regulation and activity has not been systematically addressed. We explored this issue using Saccharomyces cerevisiae, a well defined model system for the study of SFK regulation. Previous studies have shown that ectopic expression of c-Src arrests yeast cell growth in a kinase-dependent manner. We expressed Fgr, Fyn, Hck, Lck, Lyn, and Yes as well as c-Src in yeast and found that each kinase was active and induced growth suppression. Co-expression of the negative regulatory kinase Csk suppressed SFK activity and reversed the growth-inhibitory effect. We then co-expressed each SFK with HIV-1 Nef in the presence of Csk. Nef strongly activated Hck, Lyn, and c-Src but did not detectably affect Fgr, Fyn, Lck, or Yes. Mutagenesis of the Nef PXXP motif essential for SH3 domain binding greatly reduced the effect of Nef on Hck, Lyn, and c-Src, suggesting that Nef activates these Src family members through allosteric displacement of intramolecular SH3-linker interactions. These data show that Nef selectively activates Hck, Lyn, and c-Src among SFKs, identifying these kinases as proximal effectors of Nef signaling and potential targets for anti-HIV drug discovery.Nef is an accessory protein encoded by the human (HIV-1 and HIV-2) 2 and simian immunodeficiency viruses and is an essential mediator of viral pathogenicity (1-3). Experimental deletion within the simian immunodeficiency virus nef gene reduces viral load, delays the onset of AIDS-like disease, and offers immune protection against challenge with pathogenic simian immunodeficiency virus in rhesus macaques (4, 5). Strong selective pressure has been demonstrated for a functional nef gene, because some animals infected with non-pathogenic, nef-mutant simian immunodeficiency virus show in vivo repair of the mutation and progression to AIDS-like disease (5-7). In addition, some HIV-positive individuals that fail to develop AIDS exhibit nef mutations or deletions (8 -12), supporting the hypothesis that nef is essential for efficient disease progression.Nef has no known catalytic function and is believed to promote viral pathogenicity by altering signaling pathways in infected cells through its interactions with cellular proteins. Nef affects several distinct classes of host cell proteins, including immune receptors, protein kinases, trafficking proteins, and guanine nucleotide exchange factors (13-15). Through interactions with these and other signaling proteins, Nef can affect multiple cellular processes leading to enhancement of viral replication, immune evasion, and enhanced survival in T-cells and macrophages (1, 16 -18).Protein kinases are a major class of Nef effector proteins, and members of the Src family of non-receptor protein-tyrosine kinases have been str...

C-terminal Src Kinase-homologous Kinase (CHK), a Unique Inhibitor Inactivating Multiple Active Conformations of Src Family Tyrosine Kinases

“…7 that CSK inhibits SFKs only by phosphorylating their Y T (the catalytic mechanism), whereas CHK can employ both catalytic and non-catalytic inhibitory mechanisms to suppress SFK activity. Nevertheless, we cannot conclude from our findings that CHK is more important than CSK for SFK suppression in vivo because regulation of SFKs by CHK and CSK is governed by many factors including (i) the subcellular localization of CSK, CHK, and SFKs, (ii) catalytic efficiency of CSK and CHK in phosphorylating Y T of SFKs, and (iii) interaction of SFKs with activator proteins such as HIV-1 Nef and autophosphorylated FAK (18,37). Furthermore, contributions of these factors may vary in different cell types and under different physiological states.…”

HIV-1 Proteins Preferentially Activate Anti-Inflammatory M2-Type Macrophages

“…4B, 6B). The prolinerich motif of Nef was shown to bind the Src homology 3 (SH3) domains of a subset of cellular Src family tyrosine kinases, such as Hck, Lyn, and possibly c-Src (47)(48)(49)63). Among them, the interaction with Hck is important because it causes the activation of Hck kinase activity (47)(48)(49).…”

Deciphering the Cross Talk between hnRNP K and c-Src: the c-Src Activation Domain in hnRNP K Is Distinct from a Second Interaction Site

“…6). The Nef protein of human immunodeficiency virus type 1 has been shown to activate Hck, Lyn, and c-Src but not Fgr, Fyn, Lck, or Yes (33,52). The structural determinant for the activation of Hck has been mapped to the variable loop (RT loop), which is positioned close to the conserved SH3 residues implicated in the binding of proline-rich motifs.…”