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. 2017 Aug 10;6:e27159. doi: 10.7554/eLife.27159

Figure 2. The Bag-1L:AR interaction is mediated by K231/232/279 in the BAG domain of Bag-1L.

(A, B) Mammalian one-hybrid assay in HeLa cells transfected with indicated AR domains linked to Gal4 DBD, subjected to increasing concentration of Bag-1L. The results are the mean of three independent experiments ± SEM, relative to the empty Bag-1L expression vector. Schematic representations of the AR domains are shown below. AF-1: Activation function-1; H: Hinge; DBD: DNA-binding domain; LBD: Ligand-binding domain. (C) GST pull-down with GST-Bag-1L fusion proteins harboring point mutations (as indicated) in their BAG domain and lysates from LNCaP cells. Shown below is a schematic structure of Bag-1L with the triple mutations in the BAG domain, which abolish the interaction with the AR (but have no effect on Hsp70 binding). NLS: Nuclear localization sequence; UBQ: Ubiquitin-like domain; BAG: BAG domain. (D) Co-immunoprecipitation of Bag-1L and AR in LNCaP cells stably overexpressing FLAG-, HA-tagged wild-type (WT) or BAG domain mutant Bag-1L (CMut). The IP was performed using an anti-HA-tag antibody against Bag-1L and an antibody against AR and Hsp70 to evaluate binding of these proteins to Bag-1L. Equal protein loading was confirmed by probing for expression of Bag-1L, AR, Hsp70 and β-actin.. (E) Mammalian one-hybrid assay in HeLa cells transfected with pG5ΔE4-38 luciferase, TK Renilla luciferase, pM-AR AF-1 and different Bag-1L constructs harboring a wild-type or mutant BAG domain (as indicated). The results are the mean of three independent experiments ± SEM, relative to the empty Bag-1L expression vector. (F) Mammalian two-hybrid assay in HeLa cells transfected with Gal4 DBD-AR LBD and VP16-AR-AF-1 and increasing amounts of wild-type (WT) or K231/232/279A mutant Bag-1L (CMut). The results are the mean of three independent experiments ± SEM, relative to the control Renilla luciferase. (G) Log-log plot of intensities for proteins detected in forward and reverse SILAC RIME analyses of Bag-1L WT and CMut cells, targeting BAG-1L (dark blue) or IgG (light blue). Black lines represent median IgG-RIME ratios ± 2 standard deviations. Bag-1L, Hsp70 (HSPA1) and AR are indicated in yellow and red, respectively.

Figure 2—source data 1. List of Bag-1L interactors altered by the BAG domain mutation (≥2 standard deviations of Bag-1L/IgG control RIME).
elife-27159-fig2-data1.xlsx (14.8KB, xlsx)
DOI: 10.7554/eLife.27159.015
Figure 2—source data 2. Associated functions (FuncAssociate) of Bag-1L interactors altered by the BAG domain mutation.
elife-27159-fig2-data2.xlsx (28.5KB, xlsx)
DOI: 10.7554/eLife.27159.016

Figure 2.

Figure 2—figure supplement 1. Schematic of the SPOT synthesis technology.

Figure 2—figure supplement 1.

Mutant Bag-1L sequences (alanine substitutions shown in red) spanning the entire BAG domain of Bag-1L (residues 219–345) were bound to a membrane, incubated with GST-AR τau-5 and subsequently probed by Western blotting using an anti-GST antibody. Residues within non-binding regions are indicated in red. We identified seven residues (shown in red; residue numbers are indicated) critical for the interaction with AR τau-5 within the BAG domain. Residues involved in α-helix formation are highlighted in grey.
Figure 2—figure supplement 2. Overlap between AR cistromes in wild-type and CMut Bag-1L-expressing LNCaP cells.

Figure 2—figure supplement 2.

Venn diagram of AR cistromes in wild-type (WT; red) and K231A/K232A/K279A mutant Bag-1L (CMut; blue) LNCaP cells, treated with 10 nM DHT for 4 hr. The union of binding sites is indicated.
Figure 2—figure supplement 3. Top ten GO-terms (GSEA) associated with direct AR-target genes lost in the Bag-1L CMut- compared to the wild-type Bag-1L-expressing cells.

Figure 2—figure supplement 3.

Direct AR-target genes were defined as genes with DHT-induced differential expression (p≤0.05, fold change ≥1.5) that harbor a DHT-responsive AR binding site within 50 kb of the TSS of said gene. GO-terms relevant to AR action are highlighted in red.
Figure 2—figure supplement 4. Conserved BAG domain mutations that inhibit the AR AF-1 transactivation.

Figure 2—figure supplement 4.

Top, amino acid sequence of the BAG domain of Bag-1L (residues 219–345) with evolutionarily conserved residues highlighted in red and CMut (K231A/K232A/K279A) shown in blue. Residues involved in α-helix formation are highlighted in grey. Bottom, MMTV luciferase promoter assay in HeLa cells with MMTV luciferase construct, TK Renilla luciferase and ARΔLBD (amino acids 1–682), plus the indicated BAG domain mutants. The results are the mean of three independent experiments ± SD.
Figure 2—figure supplement 5. CBCACONH data of wild-type and CMut Bag-1L.

Figure 2—figure supplement 5.

Superposition of CBCACONH of Bag-1L WT (black) and the K231/232/279A mutant (Bag-1L CMut; red). 1H 13C planes were extracted from the 3D spectrum at the indicated 15N values.
Figure 2—figure supplement 6. 15N-HSQC spectra of wild-type and CMut Bag-1L.

Figure 2—figure supplement 6.

Superposition of 15N-HSQC spectra of the BAG domain of Bag-1L WT (black) and the K231/232/279A mutant (Bag-1L CMut; red).