Hsp70 cochaperones HspBP1 and BAG-1M differentially regulate steroid hormone receptor function

Abstract

Hsp70 binding protein 1 (HspBP1) and Bcl2-associated athanogene 1 (BAG-1), the functional orthologous nucleotide exchange factors of the heat shock protein 70 kilodalton (Hsc70/Hsp70) chaperones, catalyze the release of ADP from Hsp70 while inducing different conformational changes of the ATPase domain of Hsp70. An appropriate exchange rate of ADP/ATP is crucial for chaperone-dependent protein folding processes. Among Hsp70 client proteins are steroid receptors such as the glucocorticoid receptor (GR), the mineralocorticoid receptor (MR), and the androgen receptor (AR). BAG-1 diversely affects steroid receptor activity, while to date the influence of HspBP1 on steroid receptor function is mostly unknown. Here, we compared the influence of HspBP1 and BAG-1M on Hsp70-mediated steroid receptor folding complexes and steroid receptor activity. Coimmunoprecipitation studies indicated preferential binding of Hsp40 and the steroid receptors to BAG-1M as compared to HspBP1. Furthermore, Hsp70 binding to the ligand-binding domain of GR was reduced in the presence of HspBP1 but not in the presence of BAG-1M as shown by pull-down assays. Reporter gene experiments revealed an inhibitory effect on GR, MR, and AR at a wide range of HspBP1 protein levels and at hormone concentrations at or approaching saturation. BAG-1M exhibited a transition from stimulatory effects at low BAG-1M levels to inhibitory effects at higher BAG-1M levels. Overall, BAG-1M and HspBP1 had differential impacts on the dynamic composition of steroid receptor folding complexes and on receptor function with important implications for steroid receptor physiology.

Figure 1. HspBP1 and BAG-1M favor differently composed Hsp70-steroid receptor complexes.

HEK293 cells were transiently transfected with the expression plasmids of Flag-tagged wild-type or mutant, Hsp70 binding-impaired BAG-1M or HspBP1, together with 2 µg plasmid expressing either HA-tagged GR, MR, or AR. After immunoprecipitation with anti-Flag agarose, the co-precipitated steroid receptors and endogenous target proteins were analyzed by Western blot. (A–C) Representative Western blots of the input control (lysate) and the co-precipitated proteins are indicated for the experiments with GR (A), MR (B), and AR (C). The precipitated nucleotide exchange factors were visualized by colloidal Coomassie (respective lower panels). (D, E) Comparison of Hsc70/Hsp70 (D) or steroid receptor (E) binding to wild-type and mutant HspBP1 and BAG-1M. The quantity of binding to BAG-1M was used as reference and set to 100%. Shown are the mean values (+SEM, N = 3). (E) In the case of AR, a long (l) and a short (s) form were quantified separately. $ denotes not detectable binding.

Figure 2. HspBP1 reduces Hsc70 binding to GR-LBD.

Using chaperone-binding (pull-down) assays, the effect of HspBP1 and BAG-1M on Hsc70 binding to immobilized GR-LBD was analyzed with purified NEFs and in vitro translated and labeled Hsc70 in the presence or absence of ATP. Representative images of labeled Hsc70 (top, input; bottom, co-precipitation) and immuno-detected GR-LBD (after elution) are shown. The bar graph indicates the mean values of bound Hsc70 (+SEM; N = 4). The mean value of bound Hsc70 in the control sample absence of ATP) was set to 1.

Figure 3. Effects of HspBP1 and BAG-1M on GR transactivation.

The impact of HspBP1 and BAG-1M on GR function was assessed by reporter gene experiments in Cos-7 cells. Control and reporter plasmids were transfected together with plasmids expressing GR and wild-type or mutant HspBP1 or BAG-1M at the indicated amounts. 24 h after transfection, cells were treated with hormones at the indicated concentrations. In the left panel, the vector control (i.e. “empty” expression vector) was set to 100% (dashed line). In the right panel, the vector control at the highest concentration of hormone was arbitrarily set to 100%. Error bars indicate the standard error of the mean (+SEM) of four independent experiments performed in triplicates (A). Symbols indicate significant differences to vector control for BAG-1M (+), BAG1M_mut ($), HspBP1 (*), and HspBP1_mut (#), with p<0.05, 0.01, 0.001 for one, two or three symbols, respectively. Representative Western controls are shown in B and C for the indicated conditions.

Figure 4. Effects of HspBP1 and BAG-1M on MR transactivation.

The impact of HspBP1 and BAG-1M on GR function was assessed by reporter gene experiments in Cos-7 cells. Control and reporter plasmids were transfected together with plasmids expressing MR and wild-type or mutant HspBP1 or BAG-1M at the indicated amounts. 24 h after transfection, cells were treated with hormones at the indicated concentrations. In the left panel, the vector control (i.e. “empty” expression vector) was set to 100% (dashed line). In the right panel, vector control at the highest concentration of hormone was arbitrarily set to 100%. Error bars indicate the standard error of the mean (+SEM) of four independent experiments performed in triplicates (A). Symbols indicate significant differences to vector control for BAG-1M (+), BAG1M_mut ($), HspBP1 (*), and HspBP1_mut (#), with p<0.05, 0.01, 0.001 for one, two or three symbols, respectively. Representative Western blots are shown in B and C for the indicated conditions.

Figure 5. Effects of HspBP1 and BAG-1M on AR transactivation.

The impact of HspBP1 and BAG-1M on GR function was assessed by reporter gene experiments in Cos-7 cells. Control and reporter plasmids were transfected together with plasmids expressing AR and wild-type or mutant HspBP1 or BAG-1M at the indicated amounts. 24 h after transfection, cells were treated with hormones at the indicated concentrations. In the left panel, the vector control (i.e.“empty” expression vector) was set to 100% (dashed line). In the right panel, vector control at the highest concentration of hormone was arbitrarily set to 100%. Error bars indicate the standard error of the mean (+SEM) of four independent experiments performed in triplicates (A). Symbols indicate significant differences to vector control for BAG-1M (+), BAG1M_mut ($), HspBP1 (*), and HspBP1_mut (#), with p<0.05, 0.01, 0.001 for one, two or three symbols, respectively. Representative Western blots are shown in B and C for the indicated conditions.