Inhibition of transcription factor activity by nuclear compartment-associated Bcl-2.
Using a reporter gene assay in PC12, HEK293, HeLa, and NIH-3T3 cells, we show that the anti-apoptotic protein Bcl-2 significantly inhibits transcriptional activation of various transcription factors, including NF kappa B, AP1, CRE, and NFAT. A Bcl-2 mutant lacking its BH4 domain (Delta BH4) also inhibited transcription, whereas a Bcl-2 mutant lacking its transmembrane domain (Delta TM) was ineffective. Furthermore, Bcl-2 chimeric proteins containing transmembrane domains from the mitochondrial protein monoamine oxidase B (MaoB) or the endoplasmic reticulum protein cytochrome b(5) showed no effect on transcription factor activity. Subcellular localization studies showed that under conditions of transient transfection, the active Bcl-2 forms (wild type and Delta BH4) were predominantly found in the nuclear fraction, whereas the non-active forms (Delta TM, MaoB, and cytochrome b(5)) were in the non-nuclear fraction. Additionally, stably expressed Bcl-2 loses its ability to inhibit transcriptional activation and localizes predominantly to the non-nuclear fraction. Expression of FKBP38 (a chaperone that shuttles Bcl-2 to the mitochondria) removes co-expressed Bcl-2 from the nuclear fraction and reverses its effect on transcription factor activity. Finally, using an inducible gene expression system, we show that nuclear compartment-associated Bcl-2 prevents entry of NF kappa B subunits to the nucleus without affecting NF kappa B release from its cytosolic inhibitory sub-unit I kappa B alpha. These results suggest that (a) Bcl-2 suppresses transcriptional activity of multiple transcription factors; (b) Bcl-2 does not interfere with NF kappa B activation but prevents entrance of its active subunits to the nucleus; (c) membrane anchoring is required for this function of Bcl-2; and (d) association of Bcl-2 with the nuclear compartment is also necessary. We speculate that nuclear compartment-associated Bcl-2 may affect nuclear trafficking of multiple factors necessary for transcriptional activity.
Massaad
Cynthia A
CA
Department of Neuroscience and Cell Biology, University of Texas Medical Branch, Galveston, Texas 77555-1043, USA.
Portier
Bryce P
BP
Taglialatela
Giulio
G
D016475:3T3 Cells; D000818:Animals; D002467:Cell Nucleus / Q000737:chemistry* / Q000378:metabolism; D000242:Cyclic AMP; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors; D004622:Embryo, Mammalian; D015870:Gene Expression; D006367:HeLa Cells; D006801:Humans; D007668:Kidney; D051379:Mice; D016296:Mutagenesis; D016328:NF-kappa B / Q000037:antagonists & inhibitors; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins / Q000037:antagonists & inhibitors; D016716:PC12 Cells; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D051381:Rats; D020218:Response Elements / Q000187:drug effects; D013329:Structure-Activity Relationship; D022021:Tacrolimus Binding Proteins / Q000235:genetics / Q000502:physiology; D018808:Transcription Factor AP-1 / Q000037:antagonists & inhibitors; D014157:Transcription Factors / Q000037:antagonists & inhibitors*; D014158:Transcription, Genetic / Q000187:drug effects; D014162:Transfection
D004268:DNA-Binding Proteins; D016328:NF-kappa B; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D018808:Transcription Factor AP-1; D014157:Transcription Factors; D000242:Cyclic AMP; D022021:Tacrolimus Binding Proteins