Cloning and molecular characterization of mouse bcl-x in B and T lymphocytes

We report the cloning and initial characterization of the mouse homologous of the bcl-2-related gene, bcl-x. We compare these to the two major isoforms of human bcl-x that were previously identified, bcl-x(Long) (bcl-x(L)) which has death repressor activity similar to bcl-2, and bcl-x(Short) (bcl-x(S)), an alternative mRNA splice product that deletes a highly conserved domain shared by bcl-2 family members and promotes cell death in transfection studies. In addition to murine (m) bcl-x(L) and mbcl-x(S), we have cloned a novel cDNA isoform that we designate mbcl-x(ΔTM). This cDNA deletes, by means of alternative splicing, the carboxy terminal transmembrane domain of bcl-x and is predicted to be a soluble, rather than membrane-bound, protein. We found that mbcl-x mRNA is highly inducible in splenocytes stimulated with either anti-CD3 Abs or LPS/dextran sulfate, and that the major species of mbcl-x mRNA in a variety of cell lines and tissues was the x(L) isoform. Transfection of mbcl-x(L) or mbcl-x(S) into HeLa cells resulted in targeting primarily to mitochondria, whereas mbcl-x(ΔTM) localized diffusely throughout the cytosol. Overexpression of the novel ΔTM isoform in an IL-3- dependent cell line delayed the onset of apoptosis induced by growth factor withdrawal. Thus, a naturally-occurring form of mbcl-x present in lymphocytes that does not localize to the mitochondrial membrane is functional in preventing apoptotic cell death. Moreover, these data suggest that bcl-x provides a life signal in activated lymphocytes.