title: Inhibition and conformational change of SERCA3b induced by Bcl-2.
abstract: An interaction of Bcl-2 with SERCA had been documented in vitro using the SERCA1a isoform isolated from rat skeletal muscle [Dremina, E. S., Sharov, V. S., Kumar, K., Azidi, A., Michaelis, E. K., Schöneich, C. (2004) Biochem. J. 383 (361-370)]. Here, we demonstrate the interaction of Bcl-2 with the SERCA3b isoform both in vitro and in cell culture. In vitro, the interaction of Bcl-2 with SERCA3b was studied using Bcl-2∆21, a truncated form of human Bcl-2, and microsomes isolated from SERCA3b-overexpressing HEK-293 cells. For these experiments, SERCA3b was quantified by a combination of amino acid analysis and Western blotting. We observed that Bcl-2∆21 both inactivates SERCA3b and co-immunoprecipitates with SERCA3b. The incubation with Bcl-2∆21 changes the distribution of SERCA3b during sucrose density gradient centrifugation, likely as the result of Bcl-2∆21-induced conformational change of SERCA3b. When SERCA3b-overexpressing HEK-293 cells were co-transfected with Bcl-2, Bcl-2-dependent SERCA3b inactivation was observed. In these cells, Bcl-2 interaction with SERCA3b was demonstrated by co-immunoprecipitation. Furthermore, overexpression of Bcl-2 reduced fluorescein isothiocyanate (FITC) labeling of SERCA3b. Together, our data provide evidence for the interaction of Bcl-2 with SERCA3b in vitro and in cell culture, and for Bcl-2-dependent conformational and functional changes of SERCA3b.
authors.affiliation: Department of Pharmaceutical Chemistry, University of Kansas, 2095 Constant Avenue, Lawrence, KS 66047, USA.
authors.forename: Asha
authors.initials: A
authors.lastname: Hewarathna
authors.affiliation: Department of Pharmaceutical Chemistry, University of Kansas, 2095 Constant Avenue, Lawrence, KS 66047, USA.
authors.forename: Elena
authors.initials: E
authors.lastname: Dremina
authors.affiliation: Department of Pharmaceutical Chemistry, University of Kansas, 2095 Constant Avenue, Lawrence, KS 66047, USA. Electronic address: schoneic@ku.edu.
authors.forename: Christian
authors.initials: C
authors.lastname: Schöneich
mesh_terms: D057809:HEK293 Cells; D006801:Humans; D047468:Immunoprecipitation; D008861:Microsomes / Q000737:chemistry; D011487:Protein Conformation; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism*; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000378:metabolism; D017384:Sequence Deletion
chemical_list: D019253:Proto-Oncogene Proteins c-bcl-2; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; C506949:ATP2A3 protein, human
keywords: Apoptosis; Bcl-2; Ca(2+)-ATPase; Cancer; Protein-protein interaction; SERCA