FUNCTIONAL DISSECTION OF BFL-1, A BCL-2 HOMOLOG - ANTI-APOPTOSIS, ONCOGENE-COOPERATION AND CELL-PROLIFERATION ACTIVITIES

The human Bfl-1 gene codes for a 175-amino acid BCL-2 family protein t hat has an anti-apoptosis activity and is overexpressed in certain hum an epithelial and hematopoietic malignancies. Bfl-1 efficiently suppre sses apoptosis induced by the p53 tumor suppressor protein and coopera tes with a dominant nuclear oncogene, EIA, in transformation of primar y epithelial cells ill vitro. Unlike other BCL-2 family proteins, expr ession of BFL-1 permits limited cell proliferation over an extended pe riod of time when cells are induced to undergo apoptosis, We have carr ied out mutational analysis to dissect the various activities encoded by Bfl-1 and to determine the sequence requirements for these activiti es. BFL-1 shares four conserved domains, BH1, BH2, BH3 and BH4 with ot her BCL-2 family proteins. Mutations within BH1, BH2 and BH4 domains a bolish or greatly attenuate the anti-apoptotic, oncogene cooperation a nd proliferation facilitating activities of BFL-1. In contrast, a muta tion within the BH3 domain (which is essential for the activity of pro -apoptotic members of the BCL-2 family) does not significantly affect the BFL-1 functions. Although BFL-1 does not contain a well-defined C- terminal transmembrane domain, deletion of the C-terminal 24 amino aci d region (corresponding to the transmembrane domain of other BCL-2 fam ily proteins) partially reduces the various activities of BFL-1, All t he mutants defective in the anti-apoptosis activity are also defective in the oncogene cooperation activity suggesting that these two activi ties may be linked. A unique feature of BFL-1 is the presence of a Gin -rich N-terminal region that overlaps with the BH4 domain. The GIn res idues appear to be essential for the proliferation permitting activity of BFL-1. Since mutations of the GIn residues located within the BH4 domain appear to confer an extended cell survival activity in the abse nce of cell proliferation, our results suggest that BFL-1 communicates with both cell proliferation and apoptosis machineries and suggest a link between these two activities.