THE E1B 19K BCL-2-BINDING PROTEIN NIP3 IS A DIMERIC MITOCHONDRIAL PROTEIN THAT ACTIVATES APOPTOSIS

Nip3 (nineteen kD interacting protein-3) is an E1B 19K and Bcl-2 bindi ng protein of unknown function. Nip3 is detected as both a 60- and 30- kD protein in vivo and in vitro and exhibits strong homologous interac tion in a yeast two-hybrid system indicating that it can homodimerize. Nip3 is expressed in mitochondria and a mutant (Nip3(163)) lacking th e putative transmembrane domain and COOH terminus does not dimerize or localize to mitochondria. Transient transfection of epitope-tagged Ni p3 in Rat-1 fibroblasts and MCF-7 breast carcinoma induces apoptosis w ithin 12 h while cells transfected with the Nip3(163) mutant have a no rmal phenotype, suggesting that mitochondrial localization is necessar y for induction of cell death. Nip3 overexpression increases the sensi tivity to apoptosis induced by granzyme B and topoisomerase I and II i nhibitors. After transfection, both Nip3 and Nip3(163) protein levels decrease steadily over 48 h indicating that the protein is rapidly deg raded and this occurs in the absence of cell death. Bcl-2 overexpressi on initially delays the onset of apoptosis induced by Nip3 but the res istance is completely overcome in longer periods of incubation. Nip3 p rotein levels are much higher and persist longer in Bcl-2 expressing c ells. In conclusion, Nip3 is an apoptosis-inducing dimeric mitochondri al protein that can overcome Bcl-2 suppression.