Heat shock proteins attenuate SERCA inactivation by the anti-apoptotic protein Bcl-2: possible implications for the ER Ca2+ mediated apoptosis
| Autor: | Christian Schöneich, Victor S. Sharov, Elena S. Dremina |
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| Jazyk: | angličtina |
| Rok vydání: | 2012 |
| Předmět: |
SERCA
Apoptosis In Vitro Techniques Endoplasmic Reticulum Biochemistry Article Cell Line Sarcoplasmic Reticulum Calcium-Transporting ATPases Myoblasts Mice Hsp27 Microsomes Animals Humans Protein Isoforms HSP70 Heat-Shock Proteins Muscle Skeletal Molecular Biology biology Endoplasmic reticulum HEK 293 cells STIM1 Cell Biology Inositol trisphosphate receptor Molecular biology Rats Inbred F344 Cell biology Rats Protein Transport Proto-Oncogene Proteins c-bcl-2 Microsome Unfolded protein response biology.protein Calcium |
| Popis: | We have demonstrated previously that Bcl-2 and Bcl-2Δ21, a C-terminally truncated Bcl-2 sequence, inactivate SERCA (sarcoplasmic/endoplasmic reticulum Ca2+-ATPase) 1 in isolated SR (sarcoplasmic reticulum), accompanied by a translocation from CRDs (caveolae-related domains) of the SR. In the present study, we obtained evidence for the interaction of Bcl-2 with SERCA2b in C2C12 myoblasts and HEK (human embryonic kidney)-293 cells. Bcl-2 and SERCA2b co-immunoprecipitated from lysate and microsomal fractions of Bcl-2-overexpressing cells. However, Bcl-2 overexpression resulted only in a slight translocation from the CRDs and no significant SERCA inactivation. In isolated HEK-293 cell microsomes, incubation with Bcl-2Δ21 afforded SERCA2b inactivation and some translocation. HSP (heat-shock protein) 70, HSP90, HSP27 and α-crystallin attenuated Bcl-2Δ21-dependent SERCA2b inactivation. An in vitro mechanistic study with the SERCA1 isoform shows that HSP70 (i) protects SERCA1 from the inactivation by Bcl-2Δ21, (ii) inhibits SERCA1 translocation from CRD fractions, and (iii) prevents the Bcl-2Δ21-dependent loss of FITC labelling. Our data demonstrate that the mechanism of SERCA inactivation by Bcl-2 established in vitro for the SERCA1 isoform can be extended to the main housekeeping SERCA2b isoform, and that functional interactions of SERCA2b and Bcl-2 in the cell may be modulated by HSP70 and other chaperones and stress-regulated proteins. Abbreviations: cb5, cytochrome b5; CRD, caveolae-related domain; DTT, dithiothreitol; ER, endoplasmic reticulum; GFP, green fluorescent protein; GST, glutathione transferase; HEK, human embryonic kidney; HRP, horseradish peroxidase; HSP, heat-shock protein; IP, immunoprecipitation; IP3, inositol 1,4,5-trisphosphate; IP3R, IP3 receptor; LC, liquid chromatography; MAM, mitochondria-associated membrane; MCU, mitochondrial Ca2+ uniporter; MS/MS, tandem MS; NBD, nucleotide-binding domain; SERCA, sarcoplasmic/endoplasmic reticulum Ca2+-ATPase; SR, sarcoplasmic reticulum; STE, Tris-buffered saline; UPR, unfolded protein response; WB, Western blotting |
| Databáze: | OpenAIRE |
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