Phosphorylation of BCL-2 regulates ER Ca2+ homeostasis and apoptosis

Abstract

Phosphorylation of BCL-2 within an unstructured loop inhibits its antiapoptotic effect. We found that phosphorylated BCL-2 predominantly localized to the endoplasmic reticulum (ER) and tested whether phosphorylation would control its activity at this organelle, where Ca(2+) dynamics serve as a critical control point for apoptosis. Phosphorylation greatly inhibits the ability of BCL-2 to lower [Ca(2+)](er) and protect against Ca(2+)-dependent death stimuli. Cells expressing nonphosphorylatable BCL-2(AAA) exhibited increased leak of Ca(2+) from the ER and further diminished steady-state [Ca(2+)](er) stores when compared to cells expressing BCL-2(wt). Consequently, when BCL-2 is phosphorylated, Ca(2+) discharge from the ER is increased, with a secondary increase in mitochondrial Ca(2+) uptake. We also demonstrate that phosphorylation of BCL-2 inhibits its binding to proapoptotic family members. This inhibitory mechanism manifested at the ER, where phosphorylated BCL-2 was unable to bind proapoptotic members. [Ca(2+)](er) proved coordinate with the capacity of BCL-2 to bind proapoptotic BH3-only members, further integrating the apoptotic pathway and Ca(2+) modulation. Unexpectedly, the regulation of ER Ca(2+) dynamics is a principal avenue whereby BCL-2 phosphorylation alters susceptibility to apoptosis.

Figure 1

BCL-2^AAA-expressing cells are more resistant than BCL-2^wt-expressing cells to calcium-dependent death stimuli. (A) Jurkat cells expressing either neo vector, BCL-2^wt, or BCL-2^AAA were treated as indicated, stained with annexin V, and analyzed by FACS. (B) MEFs or (C) Jurkat lines were treated in medium without serum with 30 μM arachidonic acid for the indicated times. (D) Jurkat cells were treated in growth medium for 17 h with the indicated concentrations of H₂O₂. (E) Untreated Jurkat (left panel) or MEF (right panel) cells were fractionated as described in Materials and methods, and then run on SDS–PAGE and Western blotted with the indicated antibodies.

Figure 2

BCL-2^wt lowers levels of releasable ER Ca²⁺, and nonphosphorylatable BCL-2^AAA mutants further reduces levels. (A) MEF lines stably transfected with various BCL-2 constructs were loaded with the cytosolic calcium indicator Fura-2, and pulsed with 200 nM thapsigargin (TG) in the absence of extracellular calcium. After baseline was reached, calcium was added back to a final concentration of 2 mM free [Ca²⁺]. (B) For the curves shown in (A), basal and peak cytosolic levels of calcium were measured following TG-induced release of ER stores (average of 10 experiments).

Figure 3

Mitochondria in BCL-2^AAA-expressing cells take up lower levels of calcium following release from the ER. (A) MEF lines loaded with Rhod-2 were treated with 200 nM thapsigargin, and sequential images were taken. Baseline and peak fluorescence images were selected for comparison. Complete image sequences are given in Supplementary data, Movies 1–3. (B) For the images in (A), mitochondrial areas were selected, and normalized fluorescence intensity values were measured over time (see Materials and methods). (C) MEFs loaded with Rhod-2 were permeabilized with 0.001% digitonin and exposed to 14.5 μM Ca²⁺. Ruthenium red (100 μM), an inhibitor of the mitochondrial calcium uniporter, was added where indicated.

Figure 4

Cells expressing BCL-2^AAA have a lower peak ER calcium levels, and higher calcium leak rate from ER. (A) Cells transfected with ER-targeted aequorin were calcium-depleted and processed as described in Materials and methods. While monitoring aequorin luminescence, calcium was added back to obtain the peak calcium content in ER. (B) At peak calcium, SERCA-mediated ER calcium uptake was inhibited with tBuBHQ, and the rate of leak was determined.

Figure 5

BCL-2 must bind to proapoptotic proteins to lower ER calcium, and this binding is prevented by phosphorylation. (A) RIPA extracts of Jurkat cell lines were analyzed for levels of calcium handling proteins. (B) GST-BCL-2 was treated with JNK kinase, run on SDS–PAGE, and either silver stained or Western blotted with anti-S70P antibody. (C) This protein was then mixed with purified BAX or BID protein in NP-40 buffer, captured with GSH beads, and association with BAX or BID was assessed by Western blot and quantitated by densitometry. (D) Jurkat cells were treated with aphidicolin or nocodazole for 16 h, and then whole-cell extracts were made in IP buffer, immunoprecipitated with the indicated antibody, and then assayed by Western blot. (E) Following subcellular fractionation, light membrane fractions from MEF lines were solubilized in NP-40 or CHAPS IP buffer, immunoprecipitated with anti-BCL-2 antibody, and assayed for associated BIM by Western blot. (F) Fl 5.12 lines expressing the indicated constructs were loaded with Fura-2, and peak calcium following thapsigargin treatment was measured.