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HIV-1 Nef interaction influences the ATP-binding site of the Src-family kinase, Hck

Pene-Dumitrescu, T and Shu, ST and Wales, TE and Alvarado, JJ and Shi, H and Narute, P and Moroco, JA and Yeh, JI and Engen, JR and Smithgall, TE (2012) HIV-1 Nef interaction influences the ATP-binding site of the Src-family kinase, Hck. BMC Chemical Biology, 12.

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Abstract

Background: Nef 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. Results: 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 K mssssfor ATP as well as enhanced inhibitor potency. Conclusions: These observations suggest that stable interaction with Nef may induce Src-family kinase active site conformations amenable to selective inhibitor targeting. © 2012 Pene-Dumitrescu et al; licensee BioMed Central Ltd.


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Details

Item Type: Article
Status: Published
Creators/Authors:
CreatorsEmailPitt UsernameORCID
Pene-Dumitrescu, Ttsmithga@pitt.eduTSMITHGA
Shu, STSTS68@pitt.eduSTS68
Wales, TE
Alvarado, JJjja24@pitt.eduJJA24
Shi, Hhas52@pitt.eduHAS52
Narute, P
Moroco, JA
Yeh, JI
Engen, JR
Smithgall, TE
Date: 16 March 2012
Date Type: Publication
Journal or Publication Title: BMC Chemical Biology
Volume: 12
DOI or Unique Handle: 10.1186/1472-6769-12-1
Schools and Programs: School of Medicine > Microbiology and Molecular Genetics
School of Medicine > Structural Biology
Refereed: Yes
Date Deposited: 20 Oct 2016 18:22
Last Modified: 05 Feb 2019 03:55
URI: http://d-scholarship.pitt.edu/id/eprint/29942

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