Anti-apoptotic protein Bcl-2 interacts with and destabilizes the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA).
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.
Dremina
Elena S
ES
Department of Pharmaceutical Chemistry, University of Kansas, 2095 Constant Avenue, Lawrence, KS 66047, USA.
Sharov
Victor S
VS
Kumar
Keshava
K
Zaidi
Asma
A
Michaelis
Elias K
EK
Schöneich
Christian
C
D000818:Animals; D017209:Apoptosis; D001921:Brain / Q000166:cytology / Q000201:enzymology / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D027682:Cation Transport Proteins; D003545:Cysteine / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000201:enzymology*; D006801:Humans; D018482:Muscle, Skeletal / Q000166:cytology / Q000201:enzymology / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D010084:Oxidation-Reduction; D053499:Plasma Membrane Calcium-Transporting ATPases; D011480:Protease Inhibitors / Q000494:pharmacology; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D013438:Sulfhydryl Compounds / Q000378:metabolism
D027682:Cation Transport Proteins; D011480:Protease Inhibitors; D019253:Proto-Oncogene Proteins c-bcl-2; D013438:Sulfhydryl Compounds; D053499:Plasma Membrane Calcium-Transporting ATPases; D000252:Calcium-Transporting ATPases; D003545:Cysteine; D002118:Calcium