Inhibition of transcription factor activity by nuclear compartment- associated Bcl-2

Abstract

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 NFκB, AP1, CRE, and NFAT. A Bcl-2 mutant lacking its BH4 domain (ΔBH4) also inhibited transcription, whereas a Bcl-2 mutant lacking its transmembrane domain (ΔTM) was ineffective. Furthermore, Bcl-2 cliimeric proteins containing transmembrane domains from the mitochondrial protein monoamine oxidase B (HaoB) or the endoplasmic reticulum protein cytochrome b5 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 ΔBH4) were predominantly found in the nuclear fraction, whereas the non-active forms (ΔTM, MaoB, and cytochrome b5) 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 indticible gene expression system, we show that nuclear compartment-associated Bcl-2 prevents entry of NFκB subunits to the nucleus without affecting NFκB release from its cytosolic inhibitory subunit IκBα. These results suggest that (a) Bcl-2 suppresses transcriptional activity of multiple transcription factors; (b) Bcl-2 does not interfere with NFκB 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.