Anti-apoptotic protein Bcl-2 interacts with and destabilizes the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA)

Abstract

The anti-apoptotic effect of Bcl-2 is well established, but the detailed mechanisms are unknown. In the present study, we show in vitro a direct interaction of Bcl-2 with the rat skeletal muscle SERCA (sarcoplasmic/endoplasmic reticulum Ca2+-ATPase), leading to destabilization and inactivation of the protein. Recombinant human Bcl-2D21, a truncated form of Bcl-2 with a deletion of 21 residues at the C-terminal membrane-anchoring region, was expressed and affinity-purified as a glutathione S-transferase fusion protein. Bcl-2D21 co-immunoprecipitated and specifically interacted with SERCA in an in vitro-binding assay. The original level of Bcl-2 in sarcoplasmic reticulum vesicles was very low, i.e. hardly detectable by immunoblotting with specific antibodies. The addition of Bcl-2D21 to the sarcoplasmic reticulum resulted in the inhibition of the Ca2+-ATPase activity dependent on the Bcl-2D21/SERCA molar ratio and incubation time. A complete inactivation of SERCA was observed after 2.5 h of incubation at approx. 2:1 molar ratio of Bcl-2D21 to SERCA. In contrast, Bcl-2D21 did not significantly change the activity of the plasma-membrane Ca2+-ATPase. The redox state of the single Cys158 residue in Bcl-2D21 and the presence of GSH did not affect SERCA inhibition. The interaction of Bcl-2D21 with SERCA resulted in a conformational transition of SERCA, assessed through a Bcl-2-dependent increase in SERCA thiols available for the labelling with a fluorescent reagent. This partial unfolding of SERCA did not lead to a higher sensitivity of SERCA towards oxidative inactivation. Our results suggest that the direct interaction of Bcl-2 with SERCA may be involved in the regulation of apoptotic processes in vivo through modulation of cytoplasmic and/or endoplasmic reticulum calcium levels required for the execution of apoptosis.

Figure 1. Expression and purification of Bcl-2Δ21

(A) SDS/PAGE of the protein purified from the E. coli lysate: lane 1, molecular-mass standard; lane 2, Bcl-2Δ21–GST bound to glutathione–agarose beads after 4 h incubation of beads with bacterial lysate; lane 3, purified Bcl-2Δ21 in STE buffer; and lane 4, glutathione–agarose beads with remaining GST after thrombin cleavage of the fusion proteins. (B) Analysis of purified Bcl-2Δ21 by MALDI–TOF MS.

Figure 2. Effect of Bcl-2Δ21 on SERCA activities in rat skeletal-muscle SR

(A) Time-dependent inactivation of SERCA activity. SR vesicles (0.6 mg of protein/ml) were incubated in STE buffer alone (▪) or with 19 (⋄), 38 (▾), 75 (▵) and 150 (•) μg/ml Bcl-2Δ21. At the indicated times, aliquots were withdrawn and assayed for Ca²⁺-ATPase activity as described in the Experimental section. (B) The dependence of the Ca²⁺-ATPase activity on the molar ratio of Bcl-2Δ21/SERCA. SR vesicles (0.6 mg of protein/ml) were incubated for 2.5 h at 37 °C in STE buffer with different concentrations of Bcl-2Δ21 before the measurement of Ca²⁺-ATPase activity as shown in (A).

Figure 3. Bcl-2Δ21 specifically inhibits SERCA activity in rat skeletal-muscle SR

SR vesicles (12 mg of protein/ml in isolation medium containing 0.3 M sucrose and 20 mM Mops, pH 7.0) were diluted to 0.6 mg of protein/ml and incubated either in buffer containing 120 mM KCl and 20 mM Mops, pH 7.0 (1), or in the STE buffer (pH 8.0) in the absence (2) and in the presence of 150 μg/ml Bcl-2Δ21 (3), GST (4) or BSA (5), or with 4 units/ml thrombin (6), or with the addition of a comparable volume of protein-free medium obtained from E. coli transformed with pGEX3T and expressing GST alone but following the method used for Bcl-2Δ21 purification (7). The aliquots after 2.5 h incubation were withdrawn and assayed for the Ca²⁺-ATPase activity as described in the Experimental section. The activity of control sample 1 [2.50±0.24 μmol of P_i·min⁻¹·(mg of protein)⁻¹] was set equal to unity.

Figure 4. Effect of Bcl-2Δ21 on the Ca²⁺-dependent activation of SERCA

SR (0.6 mg of protein/ml) was incubated at 37 °C for 2.5 h alone (▪) or in the presence of 75 (•) or 150 (♦) μg/ml Bcl-2Δ21 in the STE buffer. (A) Samples were analysed for Ca²⁺-ATPase activity in the presence of 1 mM EGTA and different concentrations of added CaCl₂ (0.2–4 mM) as described in the Experimental section. (B) The concentration dependences normalized to the maximal activities obtained after incubations in the absence or in the presence of 75 μg/ml Bcl-2Δ21 [2.33±0.23 or 1.42±0.12 μmol of P_i·min⁻¹·(mg of protein)⁻¹ respectively] and plotted against calculated free Ca²⁺ concentrations shown in Table 1. Broken lines illustrate the determination of the Ca²⁺ concentrations for half-maximal SERCA activation (K_0.5).

Figure 5. Proteolytic degradation of SERCA and Bcl-2Δ21 and protection by protease inhibitors

(A–C, F) Rat skeletal-muscle SR (0.6 mg/ml) was incubated in absence (samples 2–5) or presence (samples 7–10) of 1 mM PMSF. Samples without (2, 4, 7, 9) and with 75 μg/ml Bcl-2Δ21 (3, 5, 8, 10) were incubated at 37 °C for 0 (2, 3, 7, 8) or 2.5 h (4, 5, 9, 10) in the STE buffer and separated by SDS/PAGE (A), analysed for Ca²⁺-ATPase activity (B), and monitored by Western blotting with anti-SERCA1 (C) or anti-Bcl-2 (F) antibodies. In (A, C, F), lanes 1 and 6 show molecular-mass standards. Samples 2–5 and 7–10 are labelled under the respective lanes (in A, C, F) or columns (B). Bands named PP and labelled by arrows indicate proteolytic degradation products. (D) SDS/PAGE analysis of Bcl-2Δ21 (150 μg/ml in STE buffer) incubated without protease inhibitors for 1 (lane 2) and 3 h (lane 3) at 37 °C or stored at 4 °C for 1 (lane 5), 2 (lane 6) or 3 days (lane 7). Bands named PP and labelled by arrows indicate proteolytic degradation products. Lanes 1 and 4 show molecular-mass-standards. (E) MALDI–TOF spectrum of Bcl-2Δ21 after storage for 3 days at 4 °C (corresponds to lane 7 in D).

Figure 6. Effect of Bcl-2Δ21 on Ca²⁺-ATPase activities of brain SPM (A) and purified PMCA (B)

Samples were incubated at room temperature at a molar ratio of 1:2 of Bcl-2Δ21/PMCA with or without 120 nM CaM. ○ and •, CaM-stimulated activities; □ and ▪, basal activities measured without (open symbols) or with (solid symbols) Bcl-2Δ21 respectively as described in the Experimental section.

Figure 7. Association of Bcl-2 and SERCA in rat skeletal-muscle SR

(A) Lysates of SR were incubated with GST–Bcl-2Δ21 (lane 2) or GST alone (lane 1), followed by affinity purification using glutathione–agarose beads as described in the Experimental section. GST–Bcl-2Δ21 and associated proteins were subsequently resolved by SDS/PAGE and immunoblotted with anti-SERCA1 and anti-GST antibody. WB, Western blot. (B) Western-blot analysis of 5 μg of SR (lane 1) with anti-SERCA1 antibodies and of proteins immunoprecipitated from 700 μg of SR with anti-Bcl-2 antibodies (lane 2). (C) Western-blot analysis of 17 (lane 1), 35 (lane 2), 70 (lane 3) μg of SR with anti-Bcl-2 antibodies, and of protein immunoprecipitated from 700 μg of SR (lane 4) with anti-Bcl-2 antibodies.

Figure 8. Conformational transition of SERCA on incubation with Bcl-2Δ21 determined by ThioGlo1 labelling of protein free cysteine residues

Protein samples were incubated with 20 μM ThioGlo1 for 1 h at 37 °C and pH 7.4 with and without 1% SDS to assay the surface-exposed and total protein free cysteine residues respectively. Fluorescent ThioGlo1–cysteine adducts were measured in 1 ml samples at excitation and emission wavelengths of 379 and 513 nm respectively (left ordinate). DTNB titration was used for quantification of protein thiols (right ordinate). a.u., arbitrary units.