title: Posttranslational modification of Bcl-2 facilitates its proteasome-dependent degradation: molecular characterization of the involved signaling pathway.
abstract: The ratio of proapoptotic versus antiapoptotic Bcl-2 members is a critical determinant that plays a significant role in altering susceptibility to apoptosis. Therefore, a reduction of antiapoptotic protein levels in response to proximal signal transduction events may switch on the apoptotic pathway. In endothelial cells, tumor necrosis factor alpha (TNF-alpha) induces dephosphorylation and subsequent ubiquitin-dependent degradation of the antiapoptotic protein Bcl-2. Here, we investigate the role of different putative phosphorylation sites to facilitate Bcl-2 degradation. Mutation of the consensus protein kinase B/Akt site or of potential protein kinase C or cyclic AMP-dependent protein kinase sites does not affect Bcl-2 stability. In contrast, inactivation of the three consensus mitogen-activated protein (MAP) kinase sites leads to a Bcl-2 protein that is ubiquitinated and subsequently degraded by the 26S proteasome. Inactivation of these sites within Bcl-2 revealed that dephosphorylation of Ser87 appears to play a major role. A Ser-to-Ala substitution at this position results in 50% degradation, whereas replacement of Thr74 with Ala leads to 25% degradation, as assessed by pulse-chase studies. We further demonstrated that incubation with TNF-alpha induces dephosphorylation of Ser87 of Bcl-2 in intact cells. Furthermore, MAP kinase triggers phosphorylation of Bcl-2, whereas a reduction in Bcl-2 phosphorylation was observed in the presence of MAP kinase-specific phosphatases or the MAP kinase-specific inhibitor PD98059. Moreover, we show that oxidative stress mediates TNF-alpha-stimulated proteolytic degradation of Bcl-2 by reducing MAP kinase activity. Taken together, these results demonstrate a direct protective role for Bcl-2 phosphorylation by MAP kinase against apoptotic challenges to endothelial cells and other cells.
authors.affiliation: Molecular Cardiology, Department of Internal Medicine IV, University of Frankfurt, Frankfurt, Germany.
authors.forename: K
authors.initials: K
authors.lastname: Breitschopf
authors.forename: J
authors.initials: J
authors.lastname: Haendeler
authors.forename: P
authors.initials: P
authors.lastname: Malchow
authors.forename: A M
authors.initials: AM
authors.lastname: Zeiher
authors.forename: S
authors.initials: S
authors.lastname: Dimmeler
mesh_terms: D017209:Apoptosis; D002478:Cells, Cultured; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D020935:MAP Kinase Signaling System; D009097:Multienzyme Complexes / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D010766:Phosphorylation; D046988:Proteasome Endopeptidase Complex; D011499:Protein Processing, Post-Translational; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
chemical_list: D009097:Multienzyme Complexes; D019253:Proto-Oncogene Proteins c-bcl-2; D014409:Tumor Necrosis Factor-alpha; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex