Abstract
The C-terminal BAG domain is thought to play a key role in BAG-1-induced survival and proliferation by mediating protein-protein interactions, for example, with heat shock proteins HSC70 and HSP70, and with RAF-1 kinase. Here, we have identified thioflavin S (NSC71948) as a potential small-molecule chemical inhibitor of these interactions. NSC71948 inhibited the interaction of BAG-1 and HSC70 in vitro and decreased BAG-1:HSC70 and BAG-1:HSP70 binding in intact cells. NSC71948 also reduced binding between BAG-1 and RAF-1, but had no effect on the interaction between two unrelated proteins, BIM and MCL-1. NSC71948 functionally reversed the ability of BAG-1 to promote vitamin D3 receptor-mediated transactivation, an activity of BAG-1 that depends on HSC70/HSP70 binding, and reduced phosphorylation of p44/42 mitogen-activate protein kinase. NSC71948 can be used to stain amyloid fibrils; however, structurally related compounds, thioflavin T and BTA-1, had no effect on BAG-1:HSC70 binding, suggesting that structural features important for amyloid fibril binding and inhibition of BAG-1:HSC70 binding may be separable. We demonstrated that NSC71948 inhibited the growth of BAG-1 expressing human ZR-75-1 breast cancer cells and wild-type, but not BAG-1-deficient, mouse embryo fibroblasts. Taken together, these data suggest that NSC71948 may be a useful molecule to investigate the functional significance of BAG-1 C-terminal protein interactions. However, it is important to recognize that NSC71948 may exert additional “off-target” effects. Inhibition of BAG-1 function may be an attractive strategy to inhibit the growth of BAG-1-overexpressing cancers, and further screens of additional compound collections may be warranted.
- BAG-1, Bcl-2 associated athanogene
- ABT-737, N-{4-[4-(4′-chloro-biphenyl-2-ylmethyl)-piperazin-1-yl]-benzoyl}-4-(3-dimethylamino-1-phenylsulfanylmethyl-propylamino)-3-nitro-benzenesulfonamide. BTA-1, 2-(4′-methylaminophenyl)benzothiazole
- CHIP, carboxy terminus of Hsc70 interacting protein
- ERK1/2, extracellular signal-regulated kinase 1/2
- NHR, nuclear hormone receptors
- NCI-DTP, National Cancer Institute Developmental Therapeutics Program
- DMEM, Dulbecco's modified Eagle's medium
- FCS, fetal calf serum
- MEF, mouse embryo fibroblast
- GST, glutathione S-transferase
- PBS, phosphate-buffered saline
- BSA, bovine serum albumin
- siRNA, small interfering RNA
- VDR, vitamin D receptor
- NLS, nuclear localization sequence
- ULD, ubiquitin-like domain
- thioflavin T, 4-(3,6-dimethyl-1,3-benzothiazol-3-ium-2-yl)-N,N-dimethylaniline chloride.
Footnotes
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This work was supported by the Biotechnology and Biological Sciences Research Council [Grant BB/C005783/1]; and the Breast Cancer Campaign and Cancer Research UK [Grant C2750/A2740].
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
doi:10.1124/jpet.109.153601
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ABBREVIATIONS:
- Received March 18, 2009.
- Accepted August 17, 2009.
- © 2009 by The American Society for Pharmacology and Experimental Therapeutics
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