INTERFERENCE OF BAD (BCL-XL BCL-2-ASSOCIATED DEATH PROMOTER)-INDUCED APOPTOSIS IN MAMMALIAN-CELLS BY 14-3-3 ISOFORMS AND P11/

Apoptosis and survival of diverse cell types are under hormonal contro l, but intracellular mechanisms regulating cell death are unclear. The Bcl-2/Ced-9 family of proteins contains conserved Bcl-2 homology regi ons that mediate the formation of home-or heterodimers important for e nhancing or suppressing apoptosis. Unlike most other members of the Bc l-2 family, BAD (Bcl-xL/Bcl-5 associated death promoter), a death enha ncer, has no C-terminal transmembrane domain for targeting to the oute r mitochondrial membrane and nuclear envelope. We hypothesized that BA D, in addition to binding Bcl-xL and Bcl-2, may interact with proteins outside the Bcl-2 family. Using the yeast two-hybrid system to search for BAD-binding proteins in an ovarian fusion cDNA library, we identi fied multiple cDNA clones encoding different isoforms of 14-3-3, a gro up of evolutionally conserved proteins essential for signal transducti on and cell cycle progression. Point mutation of BAD in one (S137A), b ut not the other (S113A), putative binding site found in diverse 14-3- 3 interacting proteins abolished the interaction between BAD and 14-3- 3 without affecting interactions between BAD and Bcl-2. Because the S1 37A BAD mutant presumably resembles an underphosphorylated form of BAD , we used this mutant to screen for additional BAD-interacting protein s in the yeast two-hybrid system. P11, a nerve growth factor-induced n eurite extension factor and member of the calcium-binding S-100 protei n family, interacted strongly with the mutant BAD but less effectively with the wild type protein. In Chinese hamster ovary (CHO) cells, tra nsient expression of wild type BAD or its mutants increased apoptotic cell death, which was blocked by cotransfection with the baculovirus-d erived cysteine protease inhibitor, P35. Cotransfection with 14-3-3 su ppressed apoptosis induced by wild type or the S113A mutant BAD but no t by the S137A mutant incapable of binding 14-3-3. Furthermore, cotran sfection with P11 attenuated the proapoptotic effect of both wild type BAD and the S137A mutant. For both 14-3-3 and P11, direct binding to BAD was also demonstrated in vitro. These results suggest that both 14 -3-3 and P11 may function as BAD-binding proteins to dampen its apopto tic activity. Because the 14-3-3 family of proteins could interact wit h key signaling proteins including Raf-1 kinase, protein kinase C, and phosphatidyl inositol 3 kinase, whereas P11 is an early response gene induced by the neuronal survival factor, nerve growth factor, the pre sent findings suggest that BAD plays an important role in mediating co mmunication between different signal transduction pathways regulated b y hormonal signals and the apoptotic mechanism controlled by Bcl-2 fam ily members.